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Related articles

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You can add citations and a bibliography to your Google Doc with these styles: Important: The citations tool is available in all languages, but the elements the tool generates in your document only appear in English at this time. For example: A book title you enter in the citations tool appears in the language you entered it.

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The List of References/Bibliography

The list of references or bibliography will be at the end of your assignment/research paper, and will usually have the heading References. References must be listed in alphabetical order.

Note: Ensure that each resource you have used in the text of your assignment appears on your reference list, and that they are identical in spelling and year.

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for books this would include place of publication and publisher's name - if two or more publisher locations are given, give the location listed first in the book.

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Note: Reference electronic books and online journal articles in the same way as print resources unless otherwise specified.

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Library Guides: Science Referencing: Reference list

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A quick guide to various referencing styles specific to science.

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Journal of Ethics | American Medical Association

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It is now possible to extract a cell from a 6- to 10-cell embryo (technically, a blastocyst) that has been fertilized in vitro, test the cell's DNA for any one of several thousand single-gene mutations, and then implant for gestation only those embryos that are free of the specified gene mutation. The procedure, called preimplantation genetic diagnosis (PGD), does not harm the embryo and enables those who wish to avoid having a child with a certain genetically transmitted disorder to do so without confronting the need for abortion. ¹ As pediatrician and bioethicist Jeffrey Botkin says, PDG's specific advantage is that certain "couples will experience a relative psychological benefit through PGD by discarding embryos to achieve a healthy child, as compared with the abortion of an affected fetus." ²

There is a trade-off, however, for circumventing the natural cycle of conceiving, undergoing more traditional forms of prenatal testing, aborting an affected fetus if necessary, then waiting for another natural pregnancy to occur, and starting the testing process again. That trade-off is the high cost in dollars, physical discomfort, and emotional stress of hormone treatment followed by invasive ova retrieval, in vitro fertilization, testing, and implantation. The entire process is often carried out at least twice, and, where more than 1 condition is being tested for, up to 8 attempts have been recorded ³. Because PGD presupposes in vitro fertilization (IVF), monetary cost for PGD begins where the cost of IVF ($11,000 and above per cycle) leaves off ³. So, as Botkin summarizes the potential market for PGD, a couple must be strenuously opposed to both abortion and to raising a child with the condition PGD is employed to identify. And they must have the financial resources to realize their wishes ⁴. Since Botkin's article in 1998, another indication for PGD has come on the scene: selecting a tissue-compatible sibling for a living child with a fatal disease with the intention of providing a bone marrow donor for the sick child. This particular intention or goal adds additional ethical complication to a practice that was not ethically neutral to start with.

Ethical Concerns of PGD

For all its expense and inconvenience, preimplantation genetic diagnosis does not escape the moral objections that many have to abortion. True, no fetus is destroyed, and the tested embryos are not harmed. Yet, embryos are created that the couple never intends to use, and chances are no one will "adopt" the unimplanted embryos that carry the unwanted mutation. Hence, those who think that human life, from fertilization forward, shares the moral status of living persons view PGD and abortion in the same light: both practices disregard or at least devalue the sanctity of life. The ethical position currently expressed in US judicial decisions and health policy confers progressively greater moral status on embryos, fetuses, and newborns along a continuum demarcated by developmental milestones such as appearance of the neural streak at about 14 days, development of the nervous system, fetal viability, and so on. Individuals who share this progressive view of moral status might suffer less from discarding embryos than from destroying a fetus.

Others object to PGD for the same range of ethical reasons that they oppose germline gene therapy and genetic engineering. Selective implantation prevents certain genotypes from coming into existence, thus threatening genetic diversity and discriminating against those with disabilities; it commodifies children, mocking the true meaning of parenting and jeopardizing the parent-child relationship; and it deprives people of the opportunity for personal and moral growth that can be realized from making the most of what "nature" bestowed upon them.

The first of these objections-curtailing genetic diversity-has been voiced by advocates for those with disabilities. Spokespersons from this camp argue that elaborate, expensive, and unnatural procedures for selecting embryos without serious genetic mutations conveys the message that people with disabilities are less highly valued than those without. The majority of bioethicists, while acknowledging that this claim of prejudicial devaluing has merit, contend that the possible psychological harm done to persons with disabilities does not justify restricting the reproductive freedom of couples who wish to reduce their risk of having a child with a disability.

The technique can, at present, be used to select embryos by sex and may, in the future, be able to allow selection for certain other non-health-related traits. These real and potential applications raise a host of ethical concerns beyond the possibly prejudicial avoidance of offspring with disabilities. Sex selection because of a sex-linked disorder is generally acceptable to those who accept PGD at all, but many oppose selecting sex for "family balancing" or because parents prefer to have a son rather than a daughter, or vice versa. Trait selection for talent, personality, or non-health-related physical attributes comes under greater ethical scrutiny, even though it is not technically possible at this time. The "chosen child" faces a determinism more forceful and rigid than genes, according to David King: parental determination that the child fulfill the intention or talent or skills it was selected to embody ⁵. King also worries about "opening the human gene pool to the winds of social market forces," that is, the transient, culturally influenced concepts of the ideal or perfect person ⁶.

PGD To Rescue Siblings

In recent and highly publicized applications, parents of children with fatal disorders have undergone IVF and PGD to select embryos that can provide bone marrow transplants for the sick child. This use of PGD has a less-exacting predecessor. In the 1980s, several families conceived offspring with the hope of having a child that could provide bone marrow for his or her sibling. In the famous Ayala case, the gamble paid off, and Marissa Eve Ayala rescued her older sister from leukemia. PGD improves the odds for success by ensuring that the implanted embryo is disease free and compatible. Some parents, in their endeavor to save a sick child, are willing to endure the repeated hormone injections, ova extractions, testing, and implantation that the procedure requires. But the parameters for producing a suitable embryo are twice as stringent as for those who seek a "merely" healthy child. The "sibling-saving" embryo must be, first, disease free and, second, histo-compatible. And, of course, a clock is running, ticking away the life of the sick child.

In the 2 cases recently written about for the New York Times by Lisa Belkin, each family had a child with Fanconi anemia, a recessive inherited disorder that causes bone marrow failure ³. Together, the mothers underwent a total of 14 IVF cycles within 2 ½ years. Some ova retrieval and fertilization produced no embryos that were both healthy (no Fanconi mutation) and compatible with the existing child's tissue. Six times implantation of healthy, matching embryos, failed to result in pregnancy, and twice pregnancies resulted in miscarriages. One mother alone had 353 hormone injections over the 2 ½-year period. In the end, 1 of the PGD pregnancies resulted in a healthy, tissue-compatible embryo and in a sibling-saving child. The rescued child is confined to her home, where her teacher and ballet instructor come to deliver their lessons and where her mother feeds her through a stomach tube 4 times a day. Her anemia is under control.

Conclusion

In addition to the unimaginable distress and pain that these families endured (and sometimes manifested: 1 of the 2 sets of parents admittedly badgered the researcher, whose own wife was dying from breast cancer and who was under investigations for using federal funding for embryo research); and the ethical issues raised by any application of PGD, this application forces the question of whether it is moral to use one person as the means to save another. The answer to the question, which has been asked since the Ayala case, is usually, "We love our baby for who she is. She is not only a rescuer or an instrument. We love her dearly." But after observing the desperation of the 2 sets of parents with fatally ill children, Belkin asks,

If society gives its blessing to the use of one child to save another, then what would prevent couples . . . from aborting when the process was far enough along that the cord blood could be retrieved? Or what would prevent couples whose child needed a new kidney from waiting until the fetal kidney was large enough, then terminating the pregnancy and salvaging the organs ³?

The hope PGD offers comes at a high price to all involved: to the parents; to the sick child, whose parents are constantly undergoing distressful clinical operations; to (in the case Belkin recounts) the researchers and clinicians; and to the child-savior itself. Considering these costs and the far-from-settled ethical problems PGD poses, efforts to improve the success rate of tissue transplants from non-sibling donors may be a worthier research endeavor for the present.

Citation

Virtual Mentor. 2001;3(8):265-268.

DOI
10.1001/virtualmentor.2001.3.8.gnth1-0108.
The viewpoints expressed in this article are those of the author(s) and do not necessarily reflect the views and policies of the AMA.

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It is now possible to extract a cell from a 6- to 10-cell embryo (technically, a blastocyst) that has been fertilized in vitro, test the cell's DNA for any one of several thousand single-gene mutations, and then implant for gestation only those embryos that are free of the specified gene mutation.

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An IVF Embryo Test Aims to Prevent Miscarriages: Is It Worth It?

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The test could help ensure a successful pregnancy but still leaves hopeful parents with some difficult decisions

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Opinions for the use of PGT in a specific case

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Opinions for the use of PGT in a specific case

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With advances in screening for unborn children with Down syndrome, the future of genetic selection is now. This offers up grave questions about the value of people with Down syndrome, and of what it means to be human in the 21st century.
It's estimated that around 60 per cent of pregnancies diagnosed with Down syndrome in New Zealand are terminated. This figure is likely to go up, with the Ministry of Health looking at introducing a new, cutting-edge screening programme.

Non-Invasive-Pre-Natal Testing (NIPT) has been hailed with excitement among the medical community, since a simple blood test can screen for Down syndrome with 99 per cent accuracy. In countries where NIPT is well established, rates of babies born with Down syndrome has reduced even further. In Europe, around 92 percent of pregnancies diagnosed with Down syndrome are terminated. And in Iceland, not a single baby has been born with Down syndrome in the past five years. Genetic selection is no longer the stuff of sci-fi. It is real, and it is here.

In New Zealand, the Ministry of Health is 'currently undertaking policy work to consider the inclusion of NIPT' in its public screening programme. Already available privately in New Zealand, it appears just a matter of time before this is publically funded. Which has left the Down syndrome community 'sick and scared', according to Mike Sullivan, head of advocacy group Saving Downs.

It's hard to escape the irony that we are celebrating Disability Awareness Sunday on 18 June, while endangering the very people we claim to celebrate. Genetic selection has profound implications not just for disability in New Zealand, but for us as a society.

Meet Vincenzo

Vincenzo Vaccarino, 22, describes himself as a people person. 'I am caring, helpful, independent and hardworking,' says Vincenzo, who has Down syndrome.

He recently got engaged to his long-time girlfriend, with a proposal worthy of the silver screen. 'It was a picnic at sunset in the rose garden at our local park. I asked my friends to help me change the words of a Bruno Mars song, which they sang to Rachel. I also gave each member of our families a letter that spelt out "Will You Marry Me?", and then I knelt down and asked her to marry me.

It was such an amazing feeling when she said yes,' he recalls.

Vincenzo dreams of being a public speaker, advocating for people with Down syndrome. When it comes to the topic of termination on the basis of Down syndrome, he doesn't mince words: 'I think it is cruel. People
should consider the rights of people with Down syndrome. We have rights -human rights!'

He adds, more reflectively, 'It makes me feel bad and I feel very uncomfortable and very sad to hear that parents choose not to have babies with Down syndrome. I think people should let people be who they are, they shouldn't judge people or try to modify people.'

Vincenzo doesn't see himself as a person with a disability. 'I am proud to have Down syndrome,' he says. But adds, 'Some people do [see it as a disability]. Some people bully me.'

In the Western medical and economic model, there is an unspoken preference for people who contribute financially to society. This is often referred to as 'ableism' in the disability sector. According to Mike, this was formative in the genesis of prenatal screening, first introduced to New Zealand in 1968. 'A lot of it was driven by economics, the cost of social care for people with disabilities, and the idea that people needed to be working and paying taxes in a functional, utilitarian way,' he says.

The Medical Model

Ministry of Health policy states that 'health practitioners must respect and support any decision made by women throughout the screening process'. But disability advocates argue that there is an inherent bias in the system.

If a woman is diagnosed with a Down syndrome pregnancy, it is mandatory for her to see an obstetrician who will inform her of the risks. But there is no mandate for her to connect with a support group. 'Ninety-five per cent of what it is like to live with the condition can only be communicated by other people who have lived with the experience. If you're not mandating any social support, how is that women are ever going to be fully informed before making the decision?' asks Mike.

This means that for many women, the decision on whether to terminate is driven by fear and frightening statistics. It's widely acknowledged within the Down syndrome community that many parents have felt pressured into terminations, and Saving Downs is currently gathering testimonials to present to the Ministry of Health.

Zandra Vaccarino-Vincenzo's mother and national executive officer of the New Zealand Down Syndrome Association-agrees with Mike's concerns. 'We are extremely concerned by the prenatal experiences parents are sharing with us, as it indicates that screening is presented in ways that discriminate against people with Down syndrome.'

The medical profession currently finds itself in a situation where it can save the life of one child, while terminating the life of another. Kim Porthouse, a midwife who is also the mother of a child with Down syndrome, has experienced this up close. 'I've seen women who are 23 weeks pregnant terminating a baby with a diagnosis of Down syndrome. At the same time, if someone miscarries at 23 weeks, we'll do everything we can to save the baby.' The official age of viability in New Zealand is 20 weeks.

Beyond Eugenics

The end game of 'ableism' is 'eugenics'-a belief that humanity benefits by improving the genetic quality of the human population. We don't need to look too far in our past to see that a eugenic view has grave consequences. Eugenics was one of the ideas at the heart of the Holocaust, which saw the attempted eradication of the disabled, along with Jews, Roma ('Gypsies') and homosexuals.

We currently stand at the crossroads of history. On the one hand, we have learnt its lessons-we are more inclusive and accepting of people in all areas of life. Those living with Down syndrome have better life outcomes than ever before. In the 1980s, life expectancy was only 25, but with increased support, community inclusion and participation in mainstream education, life expectancy today is about 60.

Yet, selective termination for Down syndrome and other disabilities is modern eugenics in practice. In our culture, terminating a pregnancy based on gender or race is considered abhorrent-and rightly so. But 'that in itself is a bias,' explains Mike. 'If you don't terminate for gender, but do for Down syndrome, it is discrimination. It reinforces the view that certain types of lives are more important than others.'

Screening for autism is currently being developed. Will we begin screening out autism, just as we are beginning to understand it? This is the big black hole of genetic selection. In the future, will we screen out blindness and deafness? Unwanted facial features? An obesity gene? Will we live in a dystopian reality when only so-called 'perfect' human beings get to survive? This may seem alarmist, but we must develop a robust ethical framework to match the rapidly developing science.

And for people with Down syndrome, being screened out of existence is not alarmist, it is happening.

A hopeful alternative

The worldview of Abrahamic faith communities offers a hopeful alternative. Since the foundational belief of Christianity, Judaism and Islam is that humans are made in the image of God, these faiths give humanity an intrinsic value. Not every person can contribute equally, but every person is equally valued.

When I ask Mike what value people with Down syndrome contribute to society, he challenges the belief behind the question. 'My worldview is that people are valuable for who they are, so the question of what value they bring to the world doesn't seem like a legitimate question. You don't have to defend who you are. Everyone has their strengths and challenges.'

But it is Vincenzo who answers the question best. 'I don't understand. What do you mean?' he asks. What value another human life has is not a question we have the right to ask.

by Ingrid Barratt (c) 'War Cry' magazine, 17 June 2017, pp7-9
You can read 'War Cry' at your nearest Salvation Army church or centre, or subscribe through Salvationist Resources.

The New Zealand Down Syndrome Association - www.nzdsa.org.nz

Our Vision: People with Down syndrome are valued and equal members of their community, fulfilling their goals.
Our Mission: To work alongside families, whānau and carers to support and empower people with Down syndrome to realise their potential and aspirations through all life stages and within all communities.
Our Essence: Accept, understand, connect.

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With advances in screening for unborn children with Down syndrome, the future of genetic selection is now. This offers up grave questions about the value of people with Down syndrome, and of what it means to be human in the 21st century.

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Economic implications of having a child with genetic abnormality such as Downs syndrome.

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Economic implications of having a child with genetic abnormality such as Downs syndrome.

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A review on the motivations, decision-making factors, attitudes and experiences of couples using pre-implantation genetic testing for inherited conditions

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AbstractBACKGROUND. In pre-implantation genetic testing (PGT), fertile couples undergo IVF with genetic testing of embryos to avoid conceptions with a gene

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It all costs $$$$

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https://www.qcard.co.nz/deals/... To help with this significant financial commitment, we work to make payment as easy as possible so that you are able to plan your finances.

For IVF treatment, you will be invoiced at each of the three stages. We outline some of the payment options below - you are welcome to use the selection that suits you best at the time of treatment.

Flexible payment options include cash; direct debit and online payment with credit card. We also offer Q Card on interest-free terms. (Fees, criteria and terms apply.) You can find more information about Q Card here.

Stage 1: Medication and monitoring is invoiced when you are about to begin your individual treatment plan and must be paid before medication is issued.

Stage 2: For egg collection and embryology the invoice is due before egg collection. If your egg collection is on a weekend, remember to allow enough time for payment in advance.

Stage 3: For embryo transfer, you will be invoiced on the day of the transfer. Payment is required on the same day, so again, if the transfer is due to take place on a weekend, remember to allow enough time for payment in advance.
2 Pre-payment
You are welcome to pay up-front for Stages 1, 2 & 3, per your estimate. Some people like to do this as it's something less to think about if you are already feeling nervous on the days of egg pick up or embryo transfer. Of course, we will refund any over payment or invoice additional costs at the end of your treatment cycle.
3 Fertility Cover®
Pay a single up-front fee for up to three cycles of IVF over an 18 month period. If treatment does not result in a live birth after the three cycles, you will receive a 70% refund. You can find the details here or speak to the clinic for more details.

Important to note:

Prices are in NZ dollars and include gst. Prices are subject to change without notice (although your treatment quote is usually held for a month).

Payment can be made by cash, direct debit, online payment with credit card or Q Card.

The chance of pregnancy is dependent on many factors. There is no refund if you do not become pregnant unless you have the Fertility Cover® option (see above).

Medications cannot be refunded once they have been issued to you.

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https://www.qcard.co.nz/deals/... To help with this significant financial commitment, we work to make payment as easy as possible so that you are able to plan your finances. For IVF treatment, you will be invoiced at each of the three stages.

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Biological Implications - Childhood obesity

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Biological Implications - Childhood obesity

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A great source with some good information for societal implications, a bit for biological implications and some good ideas for opinions against.

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Kenta Nakai, Academic Editor and Tomaiuolo Rossella, Academic Editor

Abstract

Preimplantation genetic testing (PGT) has emerged as a revolutionary technique in the field of reproductive medicine, allowing for the selection and transfer of healthy embryos, thus reducing the risk of transmitting genetic diseases. However, despite remarkable advancements, the implementation of PGT faces a series of limitations and challenges that require careful consideration. This review aims to foster a comprehensive reflection on the constraints of preimplantation genetic diagnosis, encouraging a broader discussion about its utility and implications. The objective is to inform and guide medical professionals, patients, and society overall in the conscious and responsible adoption of this innovative technology, taking into account its potential benefits and the ethical and practical challenges that it presents.

Keywords: preimplantation genetic testing, biopsy, pgt-m, pgt-a, pgt-sr, limits

1. Introduction

Preimplantation genetic testing (PGT) is a diagnostic procedure that allows the analysis of embryos during the initial stages of development, enabling the selection and transfer of healthy ones into the maternal uterus, thereby reducing the risk of transmitting severe genetic disorders and enhancing the likelihood of a successful pregnancy. Moreover, it empowers couples to avoid the challenging decision of pregnancy termination in cases of embryos affected by serious genetic conditions. PGT techniques are widely used in clinical practice today and, at the same time, represent an area of significant interest, including commercial, in terms of scientific and technological research. PGT involves a multidisciplinary approach, as it is inseparable from medically assisted reproduction (MAR) techniques and facilitates the investigation of monogenic hereditary disorders (PGT-M) and numerical (PGT-A) and/or structural (PGT-SR) chromosomal abnormalities. This procedure originated in the 1990s with methods based on polymerase chain reaction (PCR), and, since then, it has developed over time with the implementation of various strategies. Such evolution has accelerated in recent years, in which molecular biology techniques applied to laboratory diagnostics have been characterized by robust technological advancements, allowing for the analysis of an increasing number of molecular targets while significantly reducing analysis times and costs.

In particular, recent advancements in human genome sequencing have led to the development of next-generation sequencing (NGS) technologies, which currently represent the gold standard for the screening of selected gene panels or extensive portions of the genome. However, the vast amount of data produced by these technologies can provide genetic information that is often challenging to interpret within a specific clinical context. Hence, despite NGS techniques surpassing traditional molecular analysis methods in terms of technological prowess, the use of the latter remains relevant in the investigation of known molecular targets, such as searching for a familial mutation or viral genome. Therefore, while it is true that technological advancements have rendered diagnostic strategies increasingly efficient, maintaining diverse reference strategies remains a superior approach. This is because some strategies might possess efficiency limitations that necessitate the implementation of custom analysis protocols tailored to the clinical case. In order to offer a comprehensive perspective to professionals and patients, as members of the Italian Network of Preimplantation Genetic Testing (N.I.D.O.), in this narrative review, we aim to address the limitations, potential, and applications of past and current PGT techniques ( Figure 1 ).

2. Biopsy Techniques

Embryonic biopsy plays a pivotal role in the ART process and in the prevention of hereditary genetic diseases. Embryos generated using assisted reproductive technologies (ART) from couples undergoing/accessing preimplantation genetic diagnosis are initially subjected to biopsy procedures to obtain an adequate number of cells for genetic screening. Genetic analysis can be performed on one of the following sample types: polar bodies (PB), blastomeres (collected during the cleavage stage), or trophectoderm cells (TE). Each of these procedures has inherent limitations and requires the opening of the zona pellucida (ZP). Over the years, various approaches have been employed, ranging from mechanical to chemical methods, culminating in the currently predominantly used laser-assisted approach. This involves the use of a laser to open the ZP, followed by the aspiration of cells using a micropipette for biopsy purposes.

Polar bodies (PBs) are products of female gametogenesis, i.e., oogenesis, not significant from a reproductive perspective, as they cannot be fertilized. PB biopsy can be performed either simultaneously, where both PBs are collected together 16 h after insemination, or sequentially, where the first PB is retrieved before insemination (4 h after oocyte retrieval) and the second after fertilization (16-18 h after insemination). The single-step approach appears more advantageous as it allows only fertilized oocytes to be analyzed, reducing analysis time and costs. While PB biopsy is the less invasive approach, on the other hand, it only provides information mainly related to maternal genetic contribution, maternal-origin meiotic aneuploidies, or maternal pathogenic variants [ 1]. This strategy, although outdated according to current best practices, remains available in countries with legal restrictions on genetic embryo evaluation and cryopreservation.

Regarding the collection of the sample from the blastomere, involving a biopsy at the cleavage stage, its main benefit would be that a biopsy performed at this stage would enable fresh transfers, as many modern genetic techniques can provide results within 24 h of sample receipt. However, despite overcoming the limitations observed with PB biopsies, it is worth considering that, in this case, only one to two blastomeres can be retrieved. This is because the embryo at this stage consists of 6-8 cells; therefore, taking more cells would result in the depletion of about 30% of the embryonic mass, consequently affecting its viability [ 2]. Furthermore, analyzing 1-2 cells does not allow the detection of potential mosaicism, representing another significant limitation of embryo biopsy at the cleavage stage.

In the case of a blastocyst on the 5th/6th day of development, the main advantage lies in the larger amount of DNA available for analysis. Since a blastocyst consists of at least a hundred cells, more cells can be retrieved compared to an embryo on the 3rd day (5-10 cells). This also increases the chances of detecting the presence of common mosaicisms in blastocysts [ 3, 4]. Another advantage is that cells are taken from the trophectoderm, which is not directly involved in fetal formation, as opposed to inner cell mass (ICM) cells. This makes the procedure less invasive for embryonic development compared to a biopsy on the 3rd day. Furthermore, due to the larger amount of analyzed DNA, the likelihood of inconclusive diagnoses is estimated to be less than 5%, and multiple analyses for different indications can be performed from the same sample after whole-genome amplification (WGA) [ 5]. Finally, some studies have demonstrated that embryos vitrified at this stage exhibit a higher survival rate compared to embryos at the cleavage stage. This allows for postponed transfer and the execution of single embryo transfer (SET) to prevent multiple pregnancies [ 6].

Timing of Biopsy

The timing of biopsy is a highly debated topic and is crucial to ensure accurate and reliable results in genetic analysis. The first approach involves creating a laser-incised hole in the ZP on day 3 and then waiting for the herniation of the blastocyst on day 5/7 [ 7]; however, for embryos that are slightly slower in development, it is preferred to wait one more day and then perform the biopsy on the 4th day [ 8]. Although widely adopted, this procedure carries the risk of herniating the ICM, and the embryo undergoes two manipulations outside the incubator. The second approach extends blastocyst culture to the stage of complete expansion. This allows simultaneous ZP opening and TE cell retrieval, while intervening only once on the embryo and opening the ZP far from the ICM [ 9]. The third approach combines the aforementioned methods, opening the ZP when the blastocyst is fully expanded and waiting for natural herniation. Patrizia Rubino et al., 2020, [ 10] demonstrated that the choice of biopsy protocol could impact clinical outcomes. Specifically, ZP opening during the blastocyst stage with simultaneous biopsy results in a higher survival rate after thawing compared to the approach that involves ZP opening during the cleavage stage [ 10]. Despite the limitations, embryonic biopsy remains the only tool for couples seeking to have healthy children and to prevent hereditary genetic diseases. Ongoing research and technological progress in this field could help to overcome some of the current limitations and further enhance the safety and efficacy of embryonic biopsy in ART. Following the biopsy, cells are washed and placed in a tube before being sent to the genetic laboratory for analysis.

3. Preimplantation Genetic Testing for Monogenic Disorders (PGT-M)

Preimplantation genetic tests have a broad range of applications, conceptually divisible into two main areas: inherited disorders, where alterations can be found in the parents (PGT-M and PGT-SR), and de novo conditions, i.e., not inherited, as in the case of PGT-A.

The objective of PGT-M testing is to avoid transferring embryos affected by a specific monogenic disease. This can only be achieved by selecting embryos that either do not carry the mutation or are healthy carriers (in the case of recessive diseases), as may occur in patients with a positive family or personal history for a monogenic condition. This necessitates a preliminary study tailored to each couple, involving family members.

In general, PGT-M can be applied to the diagnosis of all hereditary monogenic diseases for which the responsible gene has been identified, one or two index cases are available, and a diagnostic linkage analysis protocol can be developed. Conversely, it is not indicated in cases of large gene deletions/duplications or de novo triplet expansions since the phasing of the at-risk haplotype is not feasible.

An exceptional application of PGT-M is HLA typing, which aims to select a HLA-compatible embryo for an affected sibling in need of a hematopoietic stem cell transplant [ 5, 6, 7, 8, 9, 10, 11]. However, embryo selection based on HLA typing has sparked numerous ethical debates over the years, as well as the application of PGT in diseases that affect people when they are adults (for instance, Huntington's disease). In fact, for late-onset diseases, it is usually expected that the at-risk partner of the couple undergoing PGT has already undergone presymptomatic testing. However, in most cases, the at-risk partner may not wish to know their own genetic status but still be concerned about transmitting the mutation to their child. In these cases, one can either proceed with exclusion testing, thus avoiding presymptomatic testing for the at-risk partner, or perform PGT simultaneously with the analysis of embryonic aneuploidies, testing the at-risk partner without specific result disclosure (non-disclosure test).

Nevertheless, despite significant advancements and promising prospects, PGT-M presents challenges and limitations that necessitate a thorough analysis to optimize its clinical efficacy and address ethical and practical concerns.

3.1. Diagnostic Strategies

PGT-M always starts with the biopsy of the TE, from which 5-10 cells are collected. From there, the process can split into either target amplification or whole-genome amplification [ 12]. In the case of target amplification, a multiplex PCR can be performed, identifying both the mutation and genetic markers. With whole-genome amplification, options include multiplex PCR, single nucleotide polymorphism (SNP) array, or NGS. All these techniques are based on the principle of haplotyping, which involves determining the risk-associated haplotype linked to the mutation by observing how chosen informative markers (SNPs or STRs) segregate with the mutation. This approach helps to determine whether the embryo has inherited the risk allele or the wild-type allele. This is a genuine linkage analysis, which overcomes the limitations of working with low DNA quantities. Therefore, during preclinical work-up, the genotyping of SNP markers near the gene of interest in DNA samples from the couple and family members with a known genetic status is required [ 13, 14].

PGT-M originated from fragment length analysis using STRs, combined with minisequencing for mutation analysis. In parallel, another target technology using SNPs with real-time PCR was developed, enabling the identification of mutations and flanking SNP markers. The workflow involves real-time PCR SNP genotyping, TE biopsy and the lysis of collected cells, multiplex amplification using TaqMan assays for mutations and informative SNPs, real-time PCR amplification, TaqMan genotyping, linkage analysis, and haplotype determination. If CNV assays are added to TaqMan assays in the second step, the simultaneous detection of aneuploidies (PGT-M and PGT-A) is also possible.

Among the advantages of the target protocol is a very low allele drop out (ADO) rate, which increases with WGA instead. Additionally, diagnoses can be obtained quickly (within 4 h of biopsy) due to the short analysis time of real-time PCR compared to NGS. Concurrently, WGA started to develop, allowing the amplification of the entire genome with multiple displacement amplification (MDA), followed by the target amplification of STR markers and fragment length analysis. Following the introduction of WGA, PGT-M approaches diversified greatly. With target amplification, specific loci or gene regions are amplified, whereas, with WGA, the entire genome is amplified. This opens up the possibility to use other techniques, such as SNP array, especially karyomapping, suitable for PGT-M; targeted NGS, enriching specific gene regions linked to particular pathologies; and genome-wide NGS, with an enriched panel (5000 genes) to detect various pathologies [ 15]. The advantages of SNP array are the standardized procedures and reduced time to diagnosis, allowing concurrent PGT-M, PGT-A, and PGT-SR. However, there is a drawback in terms of higher costs and the analysis being limited to inherited pathologies, excluding de novo mutations due to the need for a reference. In particular, SNP genotyping methods are preferable to STRs for indirect analysis, as SNPs are more frequent in the genome compared to STRs and thus more informative in this context. Therefore, whenever possible, SNP analysis should be preferred over STR analysis [ 16]. The diagnostic protocol of the targeted strategy involves the direct identification of the disease-causing mutation post-WGA, along with analyzing at least two informative polymorphic markers, or three markers in cases where the causative mutation is unknown [ 17]. The relevant polymorphic markers are STRs, chosen for their informativeness. An informative single STR is equivalent to three informative SNPs, even though SNPs (biallelic) are more abundant (one SNP every 300-1000 bp), easier to interpret, and suitable for high-throughput analysis [ 14]. Subsequently, a multiplex PCR is set up using pre-made kits, and the heterozygosity of STRs is assessed via capillary electrophoresis. Since markers are chosen based on the chromosome affected by the mutation, those close to the gene of interest allow the discrimination of parental haplotypes. Informative markers (heterozygous) are selected for clinical testing on embryos. High-risk (mutant) and low-risk haplotypes are established during preclinical setup. The diagnostic protocol is optimized for the couple and family members to confirm the presence of the causative variant and define informative linked markers for PGT [ 18]. As a result, PGT-M is a laborious and costly procedure that requires extensive preliminary family study, leading to long waiting lists for couples embarking on this journey. This approach also enables molecular diagnosis while simultaneously testing for DNA contamination, relatedness, and technical artifacts [ 19, 20].

3.2. PGT-M Limitations

One of the main limitations of PGT-M is the genetic variability and complexity inherent to monogenic diseases themselves. Hereditary genetic diseases can be caused by different mutations on the same gene or mutations on different genes that lead to similar phenotypes. This genetic variability can make the accurate identification of specific disease-causing mutations challenging. Moreover, certain genetic diseases can be influenced by environmental or epigenetic factors, further complicating the association between genotype and phenotype. The challenge in addressing genetic variability in monogenic disorders is that a preimplantation diagnostic test should accurately identify the presence or absence of specific mutations in embryonic cells. The current clinical practice in PGT centers does not involve the preimplantation diagnosis of VUS. However, the array of mutations and the heterogeneity of phenotypes can impact the efficacy of such tests. Furthermore, the detection of Variants of Uncertain Significance (VUS) can further complicate result interpretation, generating uncertainty about which mutations actually represent a concrete risk in terms of developing the disease. Dealing with genetic variability and the complexity of monogenic disorders requires a combination of advanced molecular, diagnostic, and bioinformatic approaches. Identifying specific genetic variants responsible for a disease requires a deep understanding of its genetic basis and the metabolic pathways involved. Additionally, collaboration between clinical geneticists, molecular biologists, and bioinformaticians is crucial in interpreting genetic information correctly and identifying relevant mutations for PGT-M.

Another major technical limitation of PGT-M protocols is the small quantity of input samples, which is why WGA is performed following tubing and cell lysis in order to obtain a sufficient quantity of genomic DNA for one or more molecular analyses [ 20, 21]. As mentioned earlier, the gold standard for PGT-M involves an indirect analysis because the risk of allele drop out (ADO), amplification failure (AF), and contamination is very high when working with embryos, as only a few cells are available for the examination. Moreover, determining the haplotype through SNP array also requires a sample from at least a first-degree relative of the carrier partner. If unavailable, this leads to reduced SNP informativeness. Conversely, this limitation can be mitigated with a targeted protocol, although it is more time-intensive compared to NGS, which provides a greater quantity of information in less time.

Certain mutations (e.g., small deletions, duplications, or complex chromosomal rearrangements) cannot be detected and diagnosed using current analytical strategies. This has led to the introduction of new screening approaches capable of analyzing genetic markers present throughout the genome (genome-wide), thereby overcoming these limitations. The use of WGA has made it possible to apply genome-wide analysis methods to samples consisting of single or few cells [ 22, 23]. An example is karyomapping, which is based on SNP array technology and determines an individual's genotype by analyzing thousands of SNPs distributed across the genome. Each SNP is biallelic, presenting one of two possible nucleotides on each chromosome. Individually, they are less informative than STRs, which possess high allelic heterogeneity. However, a set of four SNP markers is sufficient to determine the genotypes of the parents and the index case [ 24]. Karyomapping is based on linkage analysis: by comparing the SNPs associated with the disease-causing mutation in the index case and parental chromosomes with those present in embryo cells, it is possible to identify the presence or absence of the mutation-carrying allele. Therefore, karyomapping marks a transition from a family- or disease-specific diagnostic approach to a "genome-wide" approach, applicable, in principle, to any monogenic alteration with informative SNPs. Moreover, this technique overcomes the issue of failed allele amplification in single-cell cases, distinguishing key SNPs in the embryo sample from non-key SNPs. This does not completely eliminate genotyping errors but significantly reduces their occurrence and, in most cases, leads to the identification of a set of key SNP markers with consistent results.

3.3. How Does Karyomapping Work?

SNP array is a highly dense array spotted with 300,000 SNPs. Firstly, the parents and a reference individual need to be genotyped-collectively called a trio-across hundreds of thousands of sites distributed throughout the genome. This is because karyomapping is based on genome-wide linkage analysis, targeting all 300,000 available platform SNPs. The first step identifies a set of informative SNP markers (heterozygous for one parent and homozygous for the other) for each of the four parental chromosomes. Subsequently, the allele phase for each informative SNP locus is determined, and the linkage of the parental risk alleles with the corresponding chromosomes is established. A linkage is defined with respect to the reference individual's genotype, usually an individual with a known disease state, such as an affected child or fetus from a previous pregnancy. The goal is to determine the parental origin of each chromosome in the embryo with regard to the reference genotype.

The first publication concerning karyomapping technology dates back to 2010 and was authored by Professor Handyside, the father of PGD, and colleagues [ 24]. This publication summarizes the key characteristics and execution of PGT-M karyomapping technology. Firstly, it is a genome-wide linkage analysis, a distinct advantage from certain viewpoints as it is performed on an extensive, non-target set of markers-specifically, all 300,000 available on the platform. Another distinguishing feature is its applicability at the single-cell level, necessitating a whole-genome amplification step.

Furthermore, unlike other methods, no patient-, family-, or disease-specific setup is required. In 2014, Natesan et al., 2014, [ 25] conducted a concordance analysis, assessing karyomapping's accuracy against the gold standard at that time-direct mutation analysis plus linkage analysis with flanking STR markers. This study involved 218 embryo samples from 44 PGD cycles, achieving an extremely high concordance rate of 97.7%. Discrepancies mainly arose in consanguineous families where, without direct mutation analysis, distinguishing the inheritance of the four alleles solely from haplotypes became more challenging. Another advantage is that, as no preclinical work-up is necessary, waiting times for setup, the potentially necessary acquisition of locus-specific probes, and validation on parents and family members are significantly reduced [ 25]. The WGA technology used in SNP array is multiple displacement amplification (MDA). This involves isothermal whole-genome amplification utilizing random hexamer molecules that bind to denatured DNA strands, continuously extending on the nascent strand, assisted by a phi polymerase. This generates large fragments and maintains high sequencing quality due to minimal biases. This feature is unique to karyomapping, as it is not shared by other techniques like NGS [ 26].

In conclusion, one of karyomapping's advantages is the substantial reduction in laboratory workload and waiting times for couples due to the absence of the need for preclinical work-up. Additionally, it enables the simultaneous analysis of PGT-M and PGT-A since both SNP genotyping and chromosome copy number information are obtained from raw data. Disadvantages include the high costs of equipment and consumables, the underlying algorithm not providing an all-in-one solution for molecular and chromosomal diagnosis, being limited to inherited chromosomal or monogenic abnormalities only, and the necessity of pertinent familial samples for haplotyping. Lastly, direct mutation analysis is not included in this approach.

3.4. Future Perspectives for PGT-M

Improving diagnostic capabilities and accessibility for PGT-M is a key direction for the future. This includes addressing challenges associated with de novo mutations or mutations that are still difficult to identify, such as repeat expansions. New technologies like haplotyping-by-sequencing offer the potential to obtain both genetic and chromosomal information in a single workflow. However, it is important to note that this approach still requires family members for phasing, and, currently, it may not be suitable for the detection of de novo mutations.

4. Preimplantation Genetic Testing for Aneuploidy (PGT-A)

PGT-A is indicated for couples with advanced maternal age, recurrent implantation failure, and recurrent spontaneous abortions. It aims to identify any numerical chromosomal abnormalities (aneuploidies) in embryos. The selection of the best embryo is often based on morphological evaluation, as it is an inexpensive, rapid, and non-invasive technique [ 27]. However, despite its widespread use, this technique is not proficient in detecting genetic abnormalities. To improve live birth rates with the transfer of a single embryo, the use of preimplantation genetic testing for aneuploidy (PGT-A) has significantly increased. Aneuploidy, often resulting from meiotic non-disjunction, with trisomy being the most common form, has led to PGT-A being considered a definitive tool for embryo selection based on euploidy [ 28]. The complex nature of PGT-A includes demanding biopsy procedures and subsequent genetic analysis. Performing biopsies during the cleavage stage may entail the risk of fewer embryos reaching the blastocyst stage [ 29].

The origins of PGT-A techniques (e.g., FISH) date back to the 1990s, when the first methods for the genetic analysis of embryonic cells were developed. However, during these early years, technology and genetic knowledge were still developing, limiting the accuracy in identifying chromosomal abnormalities. FISH, mainly used to screen common chromosomal aneuploidies involving chromosomes 13, 18, 21, X, and Y, utilizes chromosome-specific probes to determine the copy number of a given chromosome, or specific chromosomal regions, within the nucleus. The use of different fluorochromes allows specific probes to be marked, enabling the simultaneous visualization of different chromosomal targets within the same nucleus [ 30]. Centromeric-specific probes help to characterize chromosomes by defining their origin, while whole-chromosome-specific painting probes are used to characterize and define breakpoints of both balanced and unbalanced rearrangements [ 31]. However, due to the limitations of FISH analysis, embryonic biopsy techniques, and the high prevalence of mosaicism at this developmental stage, this technique did not consistently yield the desired results to improve pregnancy outcomes through assisted reproductive techniques.

Over the subsequent decades, advancements in genomics and molecular biology led to the rapid evolution of PGT-A methods, leading to the historical period of PGT-A 2.0 (2008-2012). During this period, the emergence of array CGH (aCGH) and SNP array marked a transitional phase where a different sample collection method was adopted, primarily focused on trophectoderm (TE) biopsy. This multicellular sample improved the diagnostic reliability. Technologically, this era represented a significant advancement by enabling the simultaneous analysis of all 24 chromosomes, a capability that the previous FISH methods lacked.

Array comparative genomic hybridization (aCGH) is a technique that, to date, has been somewhat surpassed by other methodologies. It is based on the fluorescent labeling of the DNA from the biopsied sample and reference DNA. These samples are co-hybridized on an array platform where probes with known sequences are spotted. These probes can be sourced from bacterial artificial chromosomes (BACs) or synthetic oligonucleotides specifically designed to cover certain gene regions. The workflow involves the detection of the fluorescent signal resulting from the co-hybridization of the two DNAs. The primary advantage, compared to previous techniques, is that the detection is automated through analysis software across the entire chromosome. Consequently, the key advantage of aCGH over FISH is the ability to cover the entire chromosome and all 24 chromosomes. The laboratory protocol involves whole-genome amplification as the initial step, as is the case with many other technologies, such as SNP array and NGS. The resolution limit, which is the smallest detectable fragment, is heavily dependent on the number of probes available across the chromosomes and the distance between each probe: for aCGH, the resolution limit in PGT is approximately 5-7 Mb. During the historical period in which aCGH was developed, it found extensive application and was utilized in numerous publications. In particular, a study by Capalbo et al., 2015, [ 32] compared aCGH and quantitative real-time PCR, both emerging during a period of significant technological advancement and both capable of screening all 24 chromosomes. From this study, involving the double biopsy and parallel analysis of 161 blastocysts using both technologies, 99.4% concordance was observed (remarkable, considering their differing protocols and underlying principles), with discordance of 0.6%. Consequently, they conducted a third technology validation study using SNP array for the discordant cases. Upon re-analyzing the biopsies of these embryos, they found that the reasons for the discordance were mainly associated with limitations pertaining to aCGH. These limitations include, first of all, the absence of chromosome-specific cut-offs because there is simply a balance of the two fluorescent signals, so aneuploidy cannot be modeled specifically for each chromosome; the WGA can also introduce amplification biases, which is why chromosome-specific cut-offs would be useful to establish the boundary between euploidy and aneuploidy for each chromosome.

In conclusion, aCGH enables the analysis of all 24 chromosomes, featuring automated, accurate, and shorter protocols compared to FISH. It has a good resolution limit (6-10 Mb), rendering it suitable for PGT-SR. However, its disadvantages include high costs, an inability to detect polyploidy, and a low resolution for mosaicism due to the absence of chromosome-specific cut-offs.

During the same historical period, SNP array technologies were also developed. An array is a high-density matrix containing up to several million probes, which allows the genotyping of hundreds of thousands of SNPs across all chromosomes. These SNPs are selected markers. Thus, a specific platform has a preset number of SNP markers, and, therefore, the position and number of these SNPs within the region of interest are fixed. Among the applications of SNP array, in addition to the previously mentioned ones in the PGT-M setting, there is chromosomal analysis. Extra or missing chromosomes can also be detected through genotyping data. Theoretically, monosomy and trisomy present distinct profiles in terms of allelic ratio values between the reference and alternative alleles. This is referred to as "B-allele frequency". Hence, these methods have allowed a more comprehensive evaluation of embryonic chromosomal profiles, enabling the detection of aneuploidies more accurately and thoroughly. Their introduction has significantly improved the ability to identify subtle quantitative chromosomal alterations that were not easily detectable using previous methods.

In recent years, PGT-A techniques have continued to advance thanks to the implementation of high-resolution NGS-based methods. These approaches enable even more precise genetic assessment, allowing the detailed detection of chromosomal mosaicism and other genetic variants. Additionally, bioinformatics and data analysis have played a crucial role in the accurate interpretation of genomic analysis results. In the current era (PGT-A 3.0), NGS methodologies are being increasingly utilized. One of the applications of NGS in PGT-A is non-invasive preimplantation genetic testing (niPGT-A). This technique is based on the active secretion of DNA by the embryo into culture media at every stage of development, with enhanced secretion at the blastocyst stage. Cultured media containing this secreted DNA can be collected and analyzed using NGS. The great advantage of this new preimplantation diagnostic technique compared to those used until now is that it is non-invasive, meaning that it is not necessary to take cells from the embryo to carry out the diagnosis. Therefore, this avoids any potential harm that the embryo could experience during a biopsy. This approach was first applied in 2018 to understand the origin of this type of DNA and progressed to a prospective study in 2020 involving eight centers worldwide to evaluate the concordance between embryo cultured media analysis and TE biopsy: this study demonstrated that comparing TE biopsy and day 5 cultured media analysis resulted in 81.8% informativeness and 63% concordance [ 33]. However, analyzing the same samples on day 6 yielded 100% informativeness, reaching a conclusive analysis for both TE biopsy and cultured media with 84% concordance. Results significantly improved with additional steps aimed at enhancing the non-invasive analysis performance. One such step was introduced to prevent DNA contamination, involving embryo washing on day 4 before the day 6 collection, as embryos need 40 h in culture to secrete the DNA required for genetic investigation [ 34]. The reporting of results from the niPGT-A is unique, as it does not provide a clear aneuploidy diagnosis. It operates as a prioritization test, offering a percentage of embryo euploidy. The analysis software prioritizes certain embryos over others, especially those with higher euploidy scores. Therefore, this strategy may cause the exclusion of potentially euploid embryos in PGT-A.

4.1. Advantages and Limitations of PGT-A

Thanks to the possibility of selecting euploid embryos before transfer, PGT-A provides a higher likelihood of a successful pregnancy and a reduction in spontaneous miscarriages. The chromosomal analysis performed allows physicians to transfer single embryos, reducing the risk of multiple pregnancies. Among other advantages, there are time efficiency (providing rapid results, enabling embryo transfer in a short timeframe) and psychological benefits, as it can alleviate the emotional burden on couples by providing information about embryo viability. Like any other technique, there are also limitations. One of the most significant is mosaicism. This term refers to the presence of different cell lineages within an embryo. The limitation of PGT-A in this case is its inability to consistently detect mosaicism, thereby risking the transfer of an embryo with chromosomal abnormalities. Another important limitation is the impact of the procedure on the embryo, as the removal of some cells from the embryo may determine a slight impact on its viability. Finally, other limitations of the technique include the possibility of false positives and false negatives, ethical considerations, a cost increase in addition to in vitro fertilization, and the probability of inadvertently discarding viable embryos.

4.2. Mosaicism

Mosaicism refers to the presence of two or more cellular lineages with different genetic compositions within an individual. It originates from postzygotic mitotic errors, including errors in chromosomal segregation and postzygotic mitotic trisomy/monosomy and the rescue of meiotic origin aneuploidies. The reported incidence of mosaicism reaches up to 73% in cleavage-stage embryos, is reduced to 2-50% in blastocysts, and is further decreased during pregnancy to <0.2%, estimated in newborns [ 35]. Mosaicism occurs more frequently in the early stages of embryonic development and is a potential source of error in PGT-A. When mosaicism is present in an embryo, there is a risk that the biopsy may not represent the entire embryo [ 36]. PGT-A using FISH is not capable of detecting mosaicism, but the application of next-generation technologies such as NGS or aCGH in TE biopsy has shown a high rate of mosaicism detection. Initially, most laboratories and clinics equated mosaic embryos with aneuploid embryos, and they were generally discarded. In humans, the prevalence and developmental potential of mosaic embryos remain subject to intense debate. Mosaicism is generally not detected at increased rates of in vitro fertilization treatment, suggesting that aneuploid cells in mosaic preimplantation embryos do not contribute to the genetic composition of live-born infants [ 37]. A small study on mosaic embryos demonstrated their ability to give rise to healthy live births [ 38]. Retrospective studies have concluded that mosaic embryos have reduced reproductive potential [ 39]. However, such data are influenced by selection bias. In particular, analyses do not account for the fact that mosaic embryos are transferred as a last option and, consequently, their reproductive performance is often measured in a highly selected subpopulation of women who have experienced previous implantation failures.

To summarize, the history of PGT-A techniques reflects the constant advancement of genetic technologies and the progress of knowledge in the field of human genetics. From initial limited methods to sophisticated sequencing-based techniques, PGT-A has revolutionized the approach to embryo selection, opening new possibilities for couples seeking a healthy pregnancy. However, research and innovation continue to drive the field as professionals seek to address the remaining challenges and further improve the accuracy and accessibility of PGT-A techniques.

5. Preimplantation Genetic Testing for Structural Rearrangements (PGT-SR)

Preimplantation genetic testing for structural rearrangements (PGT-SR) has emerged as a promising technology in the field of reproductive medicine, allowing couples with chromosomal rearrangements to reduce the risk of transmitting genetic abnormalities to their offspring [ 11]. The most common structural chromosomal rearrangements include inversions, deletions, and duplications, but, most notably, translocations, which can be inherited or occur de novo. The presence of such alterations significantly increases the risk of generating chromosomally imbalanced embryos, regardless of the woman's age.

Just like PGT-A, the first technique used for PGT-SR was FISH, with locus-specific probes and a resolution for small rearrangements <2 Mb. It required preclinical work-up and was time- and cost-intensive, not covering all 24 chromosomes. Today's technology allows for the evaluation of the entire chromosomal complement of the couple, thus analyzing the chromosomes involved in the imbalance [ 40]. Various methods are employed for PGT-SR, including FISH, aCGH, and NGS. This type of genetic testing does not require preclinical setup and is mainly performed on embryonic biopsies collected at the cleavage or blastocyst stage [ 41, 42]. However, as with any innovation, PGT-SR is not without limitations and challenges that require a thorough understanding to enhance its effectiveness and clinical applicability. Despite the advancement of high-resolution genetic sequencing techniques, the accuracy of diagnosing chromosomal abnormalities can vary based on the size and complexity of the rearrangements. Small chromosomal segments or intricate rearrangements might elude detection, jeopardizing the accuracy of the test and potentially leading to erroneous treatment decisions.

Variability in Structural Rearrangements

Depending on the breakpoints' positions in the involved chromosomes, the genetic consequences can vary widely. Some rearrangements might have no evident effects on the health of the future child, while others could lead to severe disabilities or pregnancy losses. The difficulty in predicting the precise clinical impact of a given rearrangement can complicate genetic counseling and reproductive decisions. As already mentioned, chromosomal rearrangements encompass different categories (such as balanced translocations, Robertsonian translocations, insertions, and inversions), each presenting a set of unique challenges in the context of PGT-SR [ 43]. They can also greatly vary in terms of complexity and the involved loci: some rearrangements might involve a limited number of chromosomes and regions, while others could encompass multiple chromosomes and chromosomal segments. Such diversity of rearrangements necessitates in-depth genetic analysis to identify the involved regions and assess the chromosome balance. The accurate identification of breakpoints in chromosomal rearrangements is crucial to understanding the extent of the involved segments and whether there is a loss or gain of genetic material. However, pinpointing breakpoints accurately can be complex, especially when involving repetitive or complex chromosomal regions. Errors in breakpoint identification can lead to the misinterpretation of PGT-SR results. Embryos with chromosomal rearrangements can display variability in the distribution of altered chromosomes within embryonic cells. Some embryos might be mosaic, with some cells carrying the rearrangement and others being normal. This variability makes accurately assessing the presence and balance of rearrangements a critical challenge, as it could influence the decision regarding embryo transferability. PGT-SR is not limited to assessing the embryos at hand, as it can also have implications for future generations. In cases where one parent is a carrier of a chromosomal rearrangement, careful evaluation of how the rearrangement can be transmitted to offspring is necessary [ 44]. Addressing variability in chromosomal rearrangements requires the use of advanced genetic analysis methodologies, such as array comparative genomic hybridization (aCGH) and next-generation sequencing (NGS). These techniques can help to accurately identify involved regions, breakpoints, and the presence of mosaicism. Additionally, involving expert geneticists in result interpretation is crucial in making informed decisions about embryo transferability.

In conclusion, variability in chromosomal rearrangements adds significant complexity to PGT-SR. The diversity of rearrangements, the challenge in identifying breakpoints, mosaic patterns, and transmission to the next generations demand thorough genetic assessment and careful planning. Furthermore, technological limitations in PGT-SR can influence result accuracy and reliability, potentially impacting couples' reproductive decisions. Tackling these challenges requires the ongoing development of genetic sequencing technologies, careful result interpretation, and clear, comprehensive communication with the involved couples. Integrating new technological approaches and validation strategies can contribute to improving the clinical effectiveness of PGT-SR and ensuring informed and conscious choices for the families involved.

6. Innovative Approaches

Over the years, generic protocols have been developed for monogenic diseases and numerical and structural chromosomal abnormalities, allowing for the integration of PGT-M, PGT-A, and PGT-SR. Among these is the "One PGT solution", which employs NGS (MDA protocol) to identify both monogenic diseases and numerical and structural chromosomal abnormalities.

Another strategy is "Haploseek", which combines the CNV protocol (for the identification of alterations in PGT-A and PGT-SR) with SNP array (mainly focused on PGT-M).

The latest approach is "TruSight One sequencing", an NGS approach that relies on whole-genome amplification (MDA) and does not require preclinical work-up because markers for approximately 5000 target genes are already included. This covers many genetic conditions, albeit with the limitation of requiring thorough validation before clinical use. Interpreting the results is also a complex process, as the analysis of the genome yields increasingly intricate information ( Figure 2 ).

7. Conclusions

Preimplantation genetic diagnosis offers a unique opportunity to prevent hereditary diseases, but it is crucial to understand and address the challenges associated with these techniques. A critical assessment of the technical, ethical, and clinical limitations of PGD is essential for the informed and responsible use of past, current, and future techniques. Naturally, all diagnostic limitations must not only be carefully discussed with the patient during genetic counseling but also disclosed in the informed consent process. Continuous innovation and interdisciplinary collaboration among scientists, clinicians, and ethical experts are mandatory in tackling such challenges and maximizing the beneficial potential of PGD in clinical practice and medical research.

Acknowledgments

We thank the Italian Network of Preimplantation Genetic Testing (N.I.D.O.) for supporting this work. Views and opinions expressed are, however, those of the authors only and do not necessarily reflect those of the European Union or European Commission. Neither the European Union nor the European Commission can be held responsible for them.

Funding Statement

This work was funded by the European Union-NextGenerationEU-NRRP M6C2-Investment: 2.1, "Enhancement and strengthening of biomedical research within the NSH", (grant number PNRR-MR1-2022-12376108).

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

References

30. Munné S., Lee A., Rosenwaks Z., Grifo J., Cohen J. Diagnosi delle principali aneuploidie cromosomiche negli embrioni preimpianto umani. Riprod. Ronzio. 1993;8:2185-2191. doi: 10.1093/oxfordjournals.humrep.a138001. [[ CrossRef] Google Scholar]

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Preimplantation Genetic Testing for Genetic Diseases: Limits and Review of Current Literature

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Preimplantation genetic testing (PGT) has emerged as a revolutionary technique in the field of reproductive medicine, allowing for the selection and transfer of healthy embryos, thus reducing the risk of transmitting genetic diseases. However, despite ...

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The use of preimplantation genetic testing for aneuploidy (PGT-A): a committee opinion

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Earn online CME credit related to this document at www.asrm.org/elearn

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Preimplantation genetic testing is a technique used to identify genetic defects in embryos created through in vitro fertilization (IVF) before pregnancy. Preimplantation genetic diagnosis (PGD) refers specifically to when one or both genetic parents has a known genetic abnormality and testing is performed on an embryo to determine if it also ...

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Abstract

Preimplantation genetic testing (PGT) is an early form of prenatal genetic diagnosis where abnormal embryos are identified, thereby allowing transfer of genetically normal embryos. This technology has become an integral part of Assisted Reproductive Technology (ART) procedures. Initial experiments with animals as early as 1890 and those in the mid and later part of the last century paved the forward path of ART and PGT. This review article covers the evolution of PGT and is a pointer toward current and fast-evolving technology, allowing scientists and doctors to better comprehend human reproduction, and ensure healthy pregnancy outcomes.

KEYWORDS: Assisted reproductive technology, preimplantation genetic diagnosis, preimplantation genetic screening, preimplantation genetic testing, fluorescence in situ hybridization, array comparative genomic hybridization, next-generation sequencing

INTRODUCTION

P reimplantation genetic testing (PGT) is an early form of prenatal genetic diagnosis where abnormal embryos are identified, and only genetically normal embryos are used for implantation. This has become an integral part of Assisted Reproductive Technology (ART) procedures.

Historically, the development of PGT technology dates back to 1890 with Walter Heape's experiments of successfully transferring embryos in the Belgian Hare doe rabbits.[ 1] Animal experiments continued through the first half of the 20 th Century. In 1935, Gregory Pincus, inspired by Heape's results, was able to culture rabbit oocytes to the metaphase stage of meiosis II. Professor Robert Edwards discovered that human oocytes required 37 hours for polar body extrusion and having timed each stage of human oocyte maturation, he led the way for human in vitro fertilization (IVF). Bob Edwards was the one who ideated PGT in the mid 60s. Edwards and Gardner in 1967, using euchrysine 2GNX vital staining technique, stained rabbit blastocyst sex chromatin.[ 2] The preparation was observed under the fluorescence microscope. As this technique was potentially mutagenic, it was not compatible with embryo transfer. Hence, in 1968, they biopsied 200-300 rabbit trophoblast cells. These cells were further stained for sex chromatin. The biopsied blastocyst was implanted into a pseudopregnant female rabbit. At full term, the sex of the fetus was confirmed anatomically and histologically.[ 3] This experiment became the basis for PGT and its application to test for genetically inherited diseases.

Steptoe and Edwards, in their Manchester Laboratory, made many attempts since 1970 onwards to establish IVF in humans. Dr. Carl Wood of the Monash IVF team in Melbourne reported the first IVF pregnancy in 1973, although it resulted in an early miscarriage. In 1976, Steptoe and Edwards published a case of ectopic pregnancy following transfer of an early blastocyst. After several failed attempts, medical history was made on July 25, 1978, with the birth of the world's first "test tube baby" Louis Brown.[ 4] The reintroduction of ovarian stimulation by Trounson et al. in 1981 was a major breakthrough that increased the chances of pregnancy in IVF.[ 5]

Although pregnancy rates improved with time, results of IVF for male factor infertility remained very low with failed fertilization occurring commonly as the sperm of these men did not have the ability to perform all the steps needed for fertilization.

To characterize the fertilization potential of human sperm, the hamster egg-human sperm penetration assay was developed.[ 6] In their paper, Li et al. demonstrated Uehara and Yanagimachi's classic work in 1976 of injection of human sperm into hamster oocytes showing sperm nuclear decondensation [ Figure 1 ].[ 7] The technique of sperm microinjection was pioneered by Hiramoto in the Sea Urchin in 1962[ 8] and by Lin in 1966 in mouse oocytes.[ 9]

The practical use of micromanipulation started in the mid 80's with zona drilling (ZD) and partial zona dissection (PZD) when the sperm count, motility, or morphology were low. Pioneering attempts of ZD on a mouse model were carried out by Gordon et al., using a micromanipulator to produce holes in the zona pellucida (ZP) of unfertilized mouse oocytes with acid Tyrode's solution.[ 10] The first attempts at ZD of human oocytes for the alleviation of male infertility resulted in fertilization, however, pregnancy did not ensue in the ten couples included in this report.[ 11]

The first live birth in the world with embryo micromanipulation techniques was reported by Ng and Bongso from Singapore, where insemination was done under the ZP.[ 12] This micro-insemination sperm transfer technique later became popularly known as subzonal injection of sperm (SUZI).[ 13] The earlier PZD technique did not give good results and was discontinued as it led to polyspermy, while SUZI gave better results and eventually led to the development of intracytoplasmic sperm injection (ICSI). Thus, by the end of the 1980's, several procedures of assisted fertilization had been developed and used where conventional IVF could not succeed. Microsurgical fertilization techniques helped to remove the barrier presented to the sperm by the ZP. Assisted hatching was pioneered by Cohen around the same time.[ 14]

To achieve fertilization in nature, the sperm has to penetrate the cumulus cells. This is followed by zona binding and penetration, egg-sperm membrane interaction, and oocyte activation. Lanzendorf initiated sperm microinjection. However, the fertilized oocyte only went up to the pronuclear stage. He, therefore, abandoned the technique.[ 15]

In 1992, Gianpiero Palermo, in Dr. André van Steirteghem's Laboratory in Brussels, created the first baby by sperm microinjection into the oocyte cytoplasm. The team called it ICSI [ Figure 2 ].[ 16] This discovery got them international acclaim.

In India, the first ICSI baby of South Asia "Luv Singh," was created by our team at Jaslok Hospital, Mumbai, in 1994.[ 17]

BIOPSY TECHNIQUES

Parallel to the development of IVF technology, many experiments were being performed on animal models for obtaining a single cell from the growing embryos in vitro for future genetic analysis. Wilton and Trounson from Australia demonstrated the technique of removal of one blastomere from cleavage-stage embryos in the mouse.[ 18] In 1988, Marilyn Monk with Audrey Muggleton-Harris from UK developed the trophectoderm biopsy technique followed by Preimplantation Genetic Diagnosis (PGD) using biochemical microassay in a mouse model for Lesch-Nyhan disease.[ 19] Yury Verlinsky's group demonstrated the use of the first polar body biopsy to check for a maternal unaffected gene.[ 20] The Figures ​Figures3a 3a - c show human gametes and embryos undergoing cleavage stage, tropehctoderm and polar body biopsy.

DEVELOPMENT OF MOLECULAR TECHNOLOGY

After the presentation of the Watson and Crick model of DNA in 1953, attempts to develop synthetic oligonucleotides and to sequence the genomic DNA were made. The technique of polymerase chain reaction (PCR) was introduced by Saiki et al. in 1985.[ 21] This was a major breakthrough for the analysis of monogenic disorders in the field of molecular biology.

FIRST SUCCESSFUL PREIMPLANTATION GENETIC DIAGNOSIS ATTEMPTS

Successful attempts in the mouse model bore fruition in 1990, when Handyside et al. reported pregnancies after carrying out PGD for sex-linked disease and X-linked mental retardation on biopsied human preimplantation embryos. The PCR technique was carried out to detect male embryos free of the X-linked disease.[ 22]

Handyside's group included Wilton and Delhanty with her Ph.D. student Griffin. They introduced the world's first PGD cases where male, female, and Turner syndrome embryos could be easily identified using the fluorescence in situ hybridization (FISH) technique. The biopsy was carried out by Handyside; the cells were fixed on the slide by Wilton and FISH analysis was carried out by Griffin et al.[ 23] Simultaneously, Munné et al., from USA, applied PGD using the FISH technique for the first time using directly labeled probes.[ 24]

With this many groups started using PGD technology for testing for aneuploidy and translocations by FISH and monogenic disorders by PCR. By 2001, Verlinsky et al. from Chicago reported the first successful PGD with human leukocyte antigen matching for a sib with Fanconi anemia by haplotype analysis.[ 25] This led to the concept of "Savior Sib." Using disease-free HLA-matched embryos for implantation, the previously affected child could be cured using the transplantation of cord stem cells, and bone marrow of the unaffected baby created free of disease by PGD.

The FISH technology was further improved using different probe mixtures for 5-12 chromosome pairs in multiple rounds. It was offered to women with advanced maternal age, with a history of recurrent abortions, implantation failures as well as inherited Robertsonian or reciprocal translocations and inversions. The main limitation of the FISH technology was that only around 5-12 pairs of chromosomes could be tested for aneuploidy from a total of 23 pairs of human chromosomes. Hence, further research was initiated for developing newer techniques which could test all chromosomes for aneuploidies using a single blastomere within 24-72 hours of the biopsy.

In 1999, two different groups, Wells et al. and Voullaire et al. demonstrated the use of Comparative Genomic Hybridization (CGH) technology on human blastomeres to check for aneuploidies of all chromosomes.[ 26, 27] In 2000, Voullaire et al. did an extensive study of 12 human embryos using CGH technique on more than 60 blastomeres. The study demonstrated the presence of partial aneuploidy as well as gain and loss of fragments of chromosomes which were not previously identified using the FISH analysis.[ 28]

Wilton's group, in 2001, successfully applied PGD by CGH in a 38-year-old female with a history of primary infertility followed by an unsuccessful attempt at IVF by using FISH on IVF-PGD embryos. After testing by CGH, only one of five embryos turned out to be normal for every chromosome. This effort resulted in the birth of a healthy female child.[ 29] Thereafter, CGH technology was offered to many couples successfully for the detection of all chromosomal aneuploidies and unbalanced translocations. The major drawback of the technology was the need for cryopreservation of embryos as several days were required for testing. The other drawback was the inability of this technology to detect triploidy or tetraploidy. Based on all these attempts and results in the first decade of this century, FISH still remained the most popular technique for the detection of aneuploidies within 24-72 hours.

In spite of these pioneering attempts, why did PGD not become popular?

Mastenbroek's group, in 2007, published a paper of a multicentric, randomized, controlled trial (RCT) where they compared three cycles of IVF with and without Preimplantation Genetic Screening (PGS) in women in the age group of 35-41 years. They showed that the on-going pregnancy rates and live birth rates were 10% lower in women undergoing PGS by FISH, compared to the non-PGS group in cases of advanced maternal age.[ 30] This publication led to less use of PGS for the next few years.

The concept of better pregnancy outcome using this technology finally picked up when Munne and other scientists demonstrated its benefits.

New terminology was developed to differentiate between aneuploidy screening and detection of monogenic disorders. Aneuploidy screening was termed as "PGS" whereas testing for monogenic disorders was termed as "PGD." The aneuploidy detection using FISH was termed as version 1 (PGS v1) whereas aneuploidy detection for all 24 chromosomes has now become version 2 (PGS v2). Recently, a new term Preimplantation Genetic Testing (PGT) has been introduced. The recently modified terminology is PGT-A for aneuploidy screening, PGT-SR for structural rearrangements (translocation or inversion), and PGT-M for monogenic disorders.

Considering the difficulties in the use of FISH technology, several groups perfected the long learning curve for PGT which included perfecting embryo biopsy techniques without harming the embryo and genetic diagnosis using different molecular techniques. Different groups studied the effect of day 3 cleavage-stage biopsy and day 5 blastocyst biopsy on embryo implantation and live birth outcomes. New methods were introduced for the detection of aneuploidy of all chromosomes within 24-48 hours.

Based on Wilton's use of CGH technology for preimplantation diagnosis for aneuploidy, Wells et al. with his team published a paper in 2008 including the use of microarray and CGH platforms for the detection of aneuploidy of all 23 chromosome pairs.[ 31] This technique was validated in 2011.[ 32] In the same year, Wells' group reported the first births after Preimplantation Genetic Diagnosis of structural chromosome abnormalities using array CGH (aCGH).[ 33]

In 2013, Scott's group published their clinical trial showing that the biopsy of cleavage-stage embryos significantly impaired implantation potential; however, trophectoderm biopsy of blastocyst did not have any negative effect on implantation [ Figure 4 ].[ 34] Capalbo et al. study showed that trophectoderm biopsy should be performed on all day 5, day 6, or day 7 blastocyst stage embryos to improve implantation outcome [ Figure 5 ].[ 35] Several authors have shown the benefits of PGT to improve implantation rates (IRs). Dahdouh et al. carried out a meta-analysis of RCTs and observational studies to see whether PGS with comprehensive chromosome screening (CCS) improved clinical IR and sustained IR (beyond 20 weeks) compared with routine care for embryo selection in IVF cycles. They concluded that PGS with the use of CCS technology increases clinical and sustained IRs, thus improving embryo selection particularly in patients with normal ovarian reserve [ Figure 6 ].[ 36] Other studies have shown an improvement in ICSI outcome with PGT.[ 37]

After 2010, several other methods were developed for CCS such as single-nucleotide polymorphism (SNP) Testing, Quantitative Real-Time PCR (QT-PCR), and next-generation sequencing (NGS). Furthermore, the concept of euploid Single Embryo Transfer (eSET) was introduced. Forman et al. compared CCS-eSET group with non-CCS with SET group and showed higher on-going pregnancy rate (55% vs. 42%) and lower miscarriage rate (11% vs. 25%) in CCS-eSET group. They also showed that, in CCS-eSET group, overall IR was also higher compared to non-CCS with SET group irrespective of maternal age [ Figure 7 ].[ 38] In 2013, several groups showed the successful use of NGS technology for PGT-A[ 39] and monogenic disorders.[ 40] NGS has become the most popular method due to the shorter testing time and cost-effectiveness. Recent studies by different groups for PGT-A outcomes comparing aCGH versus NGS showed marginally improved results with NGS with eSET.[ 41] Although there are several advantages of these new techniques for aneuploidy detection, due to the limitation of their sensitivity, FISH is still used for telomeric translocations and inversions.[ 42]

The current indications for PGT include repeated implantation failures, repeated pregnancy loss, advanced maternal and paternal age, male factor infertility, and genetic disorders in the parents including mosaicism of sex chromosomes, structural rearrangements, and monogenic genetic diseases. Scott et al. published a paper in 2013 showing the analysis of an RCT. The trial showed that with CCS and fresh blastocyst transfer, sustained IR was significantly higher in the CCS group (66%) compared to control non-CCS group (48%). It also showed a higher delivery rate per cycle in CCS group (85%) compared to control non-CCS group (68%).[ 43]

BENEFITS OF PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY (PGT-A)

Chromosomal aneuploidies are one of the major causes of infertility and maternal age-related reduced fertility potential. More than 70% of spontaneous miscarriages are due to chromosomal aneuploidies. PGT-A helps to shorten the time to a viable pregnancy by reducing the need of multiple IVF cycles. Euploid embryo transfer results in highest pregnancy rates and live birth rates reducing miscarriage risk independent of maternal age.

HOW MANY PREIMPLANTATION GENETIC TESTED EMBRYOS SHOULD BE TRANSFERRED?

Based on the recommendations given by the Practice Committee of the American Society for Reproductive Medicine (ASRM) and the Practice Committee of the Society for ART (SART) published in April 2017, single euploid cleavage-stage or blastocyst embryo should be transferred irrespective of the age group.[ 44]

However, PGT has its limitations under certain circumstances.[ 42] Subtelomeric deletions, mosaicism, small structural rearrangements, microdeletions, and microduplications may pose challenges. Furthermore, experienced laboratory personnel in IVF and genetics are important for a patient's success.

MITOCHONDRIAL DNA CONTENT

One of the new modalities for enhancing success is the evaluation of the mitochondrial DNA (mtDNA) content of the embryo.[ 45] The concept is that a high mtDNA copy number in euploid embryos is indicative of lower embryo viability and implantation.[ 46] This is still not the mainstay in the diagnosis of healthy energetic embryos. Victor et al. did not show any significant difference in mitochondrial levels in blastocysts irrespective of age, ploidy, or implantation potential.[ 47] As opposed to this, Fragouli et al. showed that no pregnancies resulted from blastocysts with elevated mtDNA levels.[ 48]

MOSAIC EMBRYO: TO TRANSFER OR NOT TO TRANSFER!

Embryonic chromosomal mosaicism is a condition in which more than one cell line is present, where one has a normal chromosomal constituent and others have abnormalities in chromosome number. It is assumed that mosaicism has adverse effects to the implantation and development of the embryo. Munné et al. reported that 41% of mosaic embryos resulted in on-going implantation. Complex mosaic blastocysts and embryos with >40% abnormal cells had a lower on-going IR than other mosaics.[ 49] Spinella et al. in their study showed that the extent of mosaicism influences the success rate of IVF.[ 50] Here, they used mosaic embryos with low aneuploidy percentage for implantation with higher chances of healthy live births compared to embryos with a higher percentage of mosaicism. Kushnir et al., from their study, concluded that there was a higher on-going pregnancy rate and a lower miscarriage rate when euploid embryos were used for implantation compared to the use of mosaic embryos. However, there was no significant difference in the on-going pregnancy rates or miscarriage rates among mosaic embryo transfers at any threshold of aneuploidy, and the degree of trophectoderm mosaicism was a poor predictor of on-going pregnancy and miscarriage.[ 51]

NEWER TECHNOLOGY IN PREIMPLANTATION GENETIC TESTING-M: KARYOMAPPING: BEYOND STANDARD NEXT-GENERATION SEQUENCING

In 2010, Alan Handyside with his group described the concept of karyomapping. It is genome-wide parental haplotyping using high-density SNP genotyping. Here, a linkage-based diagnosis is carried out for any single-gene defect. By knowing the genotyping of the parents and a close relative of known disease status, generally a previously affected child, this technology eliminates the need for customized test development. Karyomapping identifies informative loci for each of the four parental haplotypes across each chromosome and maps the inheritance of these haplotypes and the position of any crossovers in the proband as well as in the preimplantation embryos. Thus, it identifies the embryo-carrying normal chromosome copies.[ 52]

NONINVASIVE PREIMPLANTATION GENETIC TESTING TECHNIQUES

As embryo biopsy is an invasive procedure, efforts are being made to find different embryonic samples which do not require embryo biopsy. One of the novel approaches is the use of noninvasive PGS. Palini et al., in 2013, attempted isolation of cell-free DNA from blastocoel fluid (BF) for aneuploidy testing using the microarray technique.[ 53] Gianaroli et al. compared ploidy status of BF with trophectoderm cells, whole embryo, polar body, and/or blastomere and concluded that BF could be used as an alternative source for aneuploidy testing.[ 54] Lane et al. tried aneuploidy detection using DNA isolated from the spent culture medium.[ 55] Kuznyetsov et al. tried a combination of blastocyst culture-conditioned medium (BCCM) and BF to obtain sufficient embryonic DNA for whole genome amplification and accurate aneuploidy screening.[ 56] All these approaches are still under research.

OUR EXPERIENCE WITH PREIMPLANTATION GENETIC TESTING

We initiated PGT at Jaslok Hospital, Mumbai, in 1999, using the FISH technique.[ 57] Ours was the first center in India to offer PGT by FISH for various genetic disorders in the early 2000s.[ 58, 59, 60, 61] With PGT-A by FISH, our clinical pregnancy rate was 36% per patient and 28% per cycle. Our team reported the first live births in India for a Robertsonian[ 62] and reciprocal translocation,[ 63] inversion with a cryptic translocation picked up on pre-PGT-A workup[ 64] and pregnancy after PGT for a complex translocation.[ 65] Currently, we use the NGS platform to offer PGT-A. In our latest series of 197 cycles, our pregnancy rate was 40%. We have also successfully carried out PGT-M for the first time, in India, for genetic disorders such as Duchenne muscular dystrophy, neurofibromatosis, sickle cell anemia, Leigh syndrome, retinoblastoma, hereditary inclusion body myopathy, cardiac disorders, and carriers of BRCA1.[ 66] We recently reported for the first time, in India, twin babies born free of the autosomal dominant BRCA1 mutation to a woman who was a BRCA1 mutation carrier[ 67] and had familial hereditary cancer syndrome in herself and her close family. We also have several pregnancies in couples carrying mutations for beta thalassemia.

CONCLUSION

From the above review, we can conclude that PGT is a major diagnostic tool to prevent transmission of any known genetic disorder. It also helps in populations which are at high risk of having babies with certain genetic aberrations. PGT reduces the trauma of multiple failed IVF cycles, early miscarriages, and helps in cases of advanced maternal age to prevent the birth of a syndromic child. PGT-M protects the child from inherited monogenic disorders. With the concept of savior sibling, PGT-M is useful in some of the hematological disorders to cure an affected child.

PGT technology should be integrated into ART to offer the best outcomes to patients. However, this technology should be used judiciously, and its pitfalls should be understood.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Acknowledgment

The authors would like to thank the IVF and Genetics teams of FertilTree-Jaslok International Fertility Centre.

REFERENCES

18. Wilton L, Trounson AO. Viability of mouse embryos and blastomeres following biopsy of a single cell. Proceedings of the 18th Annual Conference, Australian Society for Reproductive Biology; Brisbane, Australia. 1986. [ Google Scholar]

23. Griffin D, Wilton L, Handyside A, Winston R, Delhanty J. Pregnancies following the diagnosis of sex in preimplantation embryos by fluorescent [[ in situ hybridisation. Br Med J. 1993;306:1382-3. PMC free article] [ PubMed] Google Scholar]

57. Parikh FR, Naik NJ, Uttamchandani SA, Nadkarni SG, Athalye AS, Madon PF, et al. The Indo-European Seminar cum Workshop on Advances in Human Cytogenetics. Lucknow: SGPGI; 1999. Preimplantation genetic diagnosis using the diode laser in a couple with a family history of haemophilia. [ Google Scholar]

61. Madon PF, Athalye AS, Naik NJ, Parikh FR. Preimplantation genetic diagnosis. In: Telang M, editor. Atlas of Human Assisted Reproductive Technologies. India: Jaypee; 2007. pp. 167-74. [ Google Scholar]

66. Madon PF, Athalye AS, Parikh FR. Principles and Practice of Fetal Medicine. New Delhi: Jaypee Brothers Publishers; 2016. Preimplantation genetic diagnosis; pp. 210-7. [ Google Scholar]

Articles from Journal of Human Reproductive Sciences are provided here courtesy of Wolters Kluwer -- Medknow Publications

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Preimplantation Genetic Testing: Its Evolution, Where Are We Today?

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Medical advice from Facebook, waiting years for a diagnosis, juggling dozens of specialists and even wishing it was cancer instead: these are the realities faced by the thousands of New Zealanders living with a rare disorder.

Some might be the only person in the country with their illness, but in total they amount to 300,000 Kiwis. It's comparable to the number with diabetes, but their pathway to care is much less certain.

For conditions that don't tick a box on a medical form - and perhaps have to be explained to the professionals themselves - there's not an easy pathway to care, treatment or support.

On International Rare Diseases day, marked today, Kiwis with rare disorders are campaigning to be recognised with a national framework.

Lizzie's story

Lizzie Haldane, 17, was diagnosed with a rare disorder by "the most amazing luck". Born with a catastrophic heart defect she had only a few days to live when her symptoms rang a bell for a recently returned cardiologist who had happened to be there that day.

She was stabilised, given open heart surgery at five days old, and diagnosed with the very rare condition known as 22q11.2 deletion syndrome - often simply 22q.

"He came back to Rotorua where Lizzie was born and he was on duty that weekend. If he hadn't been, Lizzie would have died," her mum, Sue, said.

"It was day three, but she probably would have died in a couple more days."

The Haldanes' story is not common: rare disorders sufferers typically wait five to seven years for a diagnosis, and endure three misdiagnoses in that time.

"It's a wonderful good luck story but that is not what we really desire for rare disorders, is to have their lives saved through luck," she said.

And as a condition affecting so few people around the world, a diagnosis of 22q deletion syndrome in 2003 means very little.

The syndrome - affecting one in 2000 to 4000 births - has 180 abnormalities, affecting everything from the heart to speech, to mental health.
Related articles
At times Lizzie has 12 different specialists - "a lot of balls in the air at any one time," Sue said.

"You become this all-singing, all-dancing, quasi-medical, educational expert for your child, stitching together a treatment plan across a myriad of specialties."

Lizzie's challenges could be separated into "hardware" and "software". Mental health difficulties - "software" - were 10 times more likely in people with 22q, but Haldane had struggled to get her daughter the support she needed.

"When Lizzie was around seven years old ... her views were catastrophic: would the world end today? Would Mt Ruapehū explode? Would she die in the night?

"Soon she was unable to go outside."

But her observations were "often been challenged or ridiculed by clinicians," she said.

Lizzie said anxiety was the biggest barrier, but it was an added challenge to explain her condition every day.

"Everyone knows about diabetes or asthma, but they don't really know about 22q," she said.

"It comes with a lot of stress and anxiety. Some things that normal people can handle, I can't."

"It's quite tough making friends. I am very lonely at the moment."

Another mother of a child with 22q said she has struggled to access mental health care for her 17-year-old son when he had a breakdown two years ago.

She has asked not to be named because of the stress of the condition on her son's wellbeing.

As he got older, his mental health deteriorated until he became so distressed at the thought of school that his mum would received 20 texts a day asking to be picked up. One morning he was "rolled into a ball" on the doorstep of the house sobbing "don't send me to school".

Although 22q had a high risk of schizophrenia, the rareness of the condition made it impossible for them to find mental health support.

"On paper his syndrome says massive mental health problems, high chances of schizophrenia," she said.

"When you've got a parent and a GP saying he needs help, on paper he should have just got help straight away."

"We shouldn't have to jump through hoops."

"It should be automatic, like saying 'you've been diagnosed with cancer, yes you're going to get cancer help'."

As well as a pathway to treatment, a framework could provide an informational hub. At the moment, parents of 22q children share advice over Facebook.

"Facebook is not good enough. It's great to have that support from parents, but we need somewhere more solid to go to for information."

"If you're already been to your GP, you've got nowhere else to go."

In the early pre-internet days of her son's diagnosis, when he was constantly sick, they "just winged it one day at a time".

Aubrey's story

East Auckland man Aubrey Christmas was diagnosed with amyloidosis in 2013, a terminal condition without a cure.

Amyloidosis caused proteins to transform into fibres and spread around the body, often triggering organ and tissue malfunction. Aubrey had a life-saving liver transplant in 2016.

Aubrey, 57, had already outlived his life expectancy, with most affected family members dying in their early 50s.

Aubrey's wife Jaime said his condition caused progressive loss of sensory and motor skills, heart problems, compromised renal and gastrointestinal function.

"He never knows what will hit him first when he wakes up."

But some of the biggest challenges were around its rarity: the lack of adequate support, awareness and access to medications.

Though data was lacking in New Zealand, they were believed to be one of five families in the country with Aubrey's specific condition - hereditary TTR amyloidosis.

Sometimes his low blood pressured caused him to be light-headed, needing to lean against the wall for support.

"People noticing me are quick to assume I am drunk and do not offer help," he said.

As Aubrey's full-time carer and the chief executive of NZ Amyloidosis Patients Association, Jaime was unable to work and felt torn between him and their four children - "constantly living on the edge and expecting the worst to happen".

After long struggling to access the medications in New Zealand that would prolong his life, a European pharmaceutical company had reached out and offered Aubrey treatment.

"Had we not advocated on our own, he would not have come this far," Jaime said.

"New Zealand is not a third-world country, but in some aspect, mimics one."

National framework needed

Aubrey and Jaime, as well as the 22q mothers, were signatories of a petition to be taken to Health select committee Chair Liz Crigg next month, calling urgently for a national framework.

Rare Disorders NZ Chief executive Lisa Foster said an official strategy would give patients a mechanism for treatment and support, as well as providing research and data around their conditions

The current New Zealand health system was "set up to fail" them, she said.

Australia launched the National Strategic Action Plan for Rare Diseases in 2020, and Taiwan recently celebrated the 20-year anniversary of their framework. The UK even had a minister for Rare Disease policy.

"New Zealanders would be shocked to learn how far behind we've fallen," Foster said.

"People are falling, sliding around in the dark, barriers at every turn."

"We protect rare animals, we protect rare buildings. Why can't we protect rare people?"

Some mothers of children with rare disorders had told her they wished their children had cancer instead.

"She was on a ward and all the other children were getting cancer packs with the beads and things and her child didn't," she said.

"The people in the ward said they felt sorry for her, because they got automatic support."

"They had a coordinator, so they have a pathway - a bit like a train on a track. The train goes along the track, they don't have to build the track as they go."

"There's a cancer action plan, there's a cancer agency, there's a cancer strategy ... we just want the same."

Rare Disorders NZ had been fighting since 2018, and in that time their funding had been reduced from 120,000 to 60,000 a year. There was no guarantee of funding after 2023.

The organisation was meeting with Health Minister Andrew Little on Monday in the hopes of co-designing a national framework.

The Health minister said the meeting was an opportunity to hear directly from Rare Disorders NZ on what their aspirations and expectations were.

"I understand people living with rare disorders can face challenges in accessing medicines, supports and services," he said.

"While the ministry does not currently have a specific rare diseases policy or framework, there is a range of work underway to improve the health and disability system."

"These pieces of work aim to improve the health and wellbeing of all New Zealanders, including people who are living with, or supporting family members with, rare disorders."

A Ministry of Health spokesperson said the ministry met regularly with Rare Disorders NZ, and was committed to conversations around improving support.

But Rare Disorders NZ Board Chair James McGoram said quarterly meetings with the ministry must have so far "fallen on deaf ears".

"There is an over-arching lack of consultation and recognition for rare disorders," he said.

Existing structures were "out of date, out of step" with international developments and the government's commitments, he said.

The pandemic had hit pause on the fight for a national framework, but also highlighted the need for one. People with rare disorders could be immuno-compromised, but not officially recognised as such.

Sue Haldane said the onset of coronavirus had been "really really scary times".

"It was very distressing that there was no plan, because we have no cohesive plan anyway for people with rare disorders," she said.

"When something like Covid happens and you've got a pandemic and by definition, this population of highly vulnerable people, there is no plan for that."

"This is Covid but there will be others... in times of disasters of any kind there is no provision for vulnerable populations like ours."

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'Sliding around in the dark': What it's like to have a rare disorder in New Zealand

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Medical advice from Facebook, waiting years for a diagnosis, juggling dozens of specialists and even wishing it was cancer instead: these are the realities...

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I recently had the pleasure of attending an Amyloidosis Alliance conference in Madrid, which brought together advocates from around the world who serve the transthyretin amyloidosis community. This includes the familial amyloid polyneuropathy (FAP) and amyloid light chain or primary amyloidosis communities. Attendees discussed the challenges with these diseases that we face in our respective countries.

As an advocate for New Zealand since 2019, I've found that, here, treatment accessibility is the biggest issue patients and caregivers face. Medicines approved by the U.S. Food and Drug Administration, such as Darzalex (daratumumab), Amvuttra (vutrisiran), Onpattro (patisiran), Tegsedi (inotersen), and Vyndaqel or Vyndamax (tafamidis meglumine or tafamidis), will likely spend many years waiting for local Medsafe approval. That wait will continue while funding approval is sought.

This long delay, through a very arduous process with no specific timeline, has caused me and other patient advocates countless headaches and heartbreak.

I say heartbreak because of the lack of empathy displayed for people diagnosed with a rare condition, as well as a continued disregard for caregivers when they scream for help.

Recommended Reading

What patients in New Zealand need

Having been a caregiver myself, I know how difficult it is not to have the necessary tools and coping mechanisms to best care for a loved one. I watched my husband, Aubrey, transform from an active sportsman into a shell of a human because of hereditary ATTR amyloidosis. He gradually vanished right in front of me.

Since his passing in May 2022, I've made it a personal mandate to highlight the shortcomings we face in New Zealand through the various platforms available to me in the hope that positive change will happen.

Apart from needing more treatments, patients also need proper wraparound support. As amyloidosis is a multisystemic disease that affects most of the critical organs in the body, a specialized network of clinicians must be involved in patient care. On top of this, patients and their caregivers should receive help that promotes and protects their mental health and psychosocial well-being.

It's a steep mountain to climb, but by voicing my experience and calling for peer support, I believe change will follow. As it stands, the CRISPR-Cas9 gene-editing clinical trials, sponsored by Intellia Therapeutics, have shown promise of a cure. New Zealand is fortunate enough to host one of the trial's sites.

Pulitzer Prize-winner Thomas L. Friedman said, " Big breakthroughs happen when what is suddenly possible meets what is desperately needed."

As long as I keep highlighting the urgent need for help in this part of the world, that help, I believe, will eventually arrive.
Note: FAP News Today is strictly a news and information website about the disease. It does not provide medical advice, diagnosis, or treatment. This content is not intended to be a substitute for professional medical advice, diagnosis, or treatment. Always seek the advice of your physician or other qualified health provider with any questions you may have regarding a medical condition. Never disregard professional medical advice or delay in seeking it because of something you have read on this website. The opinions expressed in this column are not those of FAP News Today or its parent company, Bionews, and are intended to spark discussion about issues pertaining to familial amyloid polyneuropathy.

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Advocating for more amyloidosis treatment options in New Zealand

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Columnist Jaime Christmas plans to remain an advocate to bring more amyloidosis treatment options to her home in New Zealand.

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Find out about amyloidosis, including causes and symptoms and how it is diagnosed and treated.

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What causes transthyretin amyloidosis (ATTR-CM)?

A change to the TTR gene causes familial (hereditary) ATTR-CM. In rare instances, this gene change arises in a person without a family history (a de novo mutation). Black people who were assigned male at birth are more likely to develop familial ATTR-CM.

Experts aren't sure why some people develop wild-type ATTR-CM. This type most commonly occurs in males over 65, which suggests age and sex may be risk factors.

Regardless of the underlying cause, your body starts to produce faulty TTR proteins. These abnormal proteins break apart, fold up on themselves (misfold) and form clumps of amyloid fibrils. Your blood carries these fibril clumps throughout your body, depositing them in nerves or organs like your heart. Over time, organ or nerve damage occurs.

What are the symptoms of transthyretin amyloidosis (ATTR-CM)?

Symptoms of transthyretin amyloidosis (ATTR-CM) vary depending on the type. People with wild-type ATTR-CM may not have any symptoms. If symptoms occur, they often appear after the age of 65.

Familial ATTR-CM symptoms most commonly occur for the first time in people over 50. However, symptoms can occur as early as your 20s or as late as your 80s.

Symptoms of transthyretin amyloidosis (ATTR-CM) are similar to heart failure symptoms, which include:

Shortness of breath (especially when lying down or doing minimal physical activity).

Bloated stomach.

Confusion or difficulty thinking.

Coughing or wheezing (especially when lying down).

Edema (fluid retention in your legs, ankles and feet).

Arrhythmia (irregular or fast heart rate), especially atrial fibrillation.

Heart palpitations.

Fatigue.

Dizziness or passing out.

What are the complications of transthyretin amyloidosis (ATTR-CM)?

Transthyretin amyloidosis (ATTR-CM) can lead to heart failure and atrial fibrillation (Afib), a type of arrhythmia.

Amyloid deposits that build up in the nervous system can cause:

Deposits may also affect the spine, leading to spinal stenosis. Deposits in tissues can cause tendon ruptures.

Amyloidosis is also often seen with a condition called aortic stenosis (narrowing of the aortic valve, which connects the main pumping chamber of your heart to your aorta). If you have aortic stenosis, your healthcare provider may consider testing you for amyloidosis (especially if you have other signs of the disease, such as abnormal heart rhythms or carpal tunnel syndrome).

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Transthyretin amyloidosis (ATTR-CM) is a buildup of faulty proteins (fibrils) on your heart or nerves. It can cause cardiomyopathy and heart failure.

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NZ organisation supporting patients with amyloidosis

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NZ organisation supporting patients with amyloidosis

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Amyloidosis is a group of diseases characterized by the production of abnormal proteins called amyloid. These proteins can accumulate and deposit in various organs, tissues, nerves, and other parts of the body, leading to impaired function and organ damage. It is important to note that there are different types of amyloidosis, each with its own specific treatments and considerations.

Systemic amyloidosis refers to the condition where amyloid clusters are found in multiple areas of the body simultaneously, while localized amyloidosis occurs when the deposits are limited to a specific area.

The three most common types of systemic amyloidosis are:

AL (Amyloid Light Chain) Amyloidosis : In this type, the amyloid protein is derived from abnormal plasma cells in the bone marrow. It is often associated with multiple myeloma or other plasma cell dyscrasias.

AA (Amyloid A) Amyloidosis: This type involves the deposition of amyloid protein derived from the serum amyloid A protein (SAA). It is usually associated with chronic inflammatory diseases, such as rheumatoid arthritis or inflammatory bowel disease.

ATTR (Transthyretin Amyloidosis): Transthyretin (TTR) protein is the source of amyloid in ATTR amyloidosis. It can be hereditary , resulting from genetic mutations, or acquired through the aging process.

Apart from these, there are other types of amyloidosis as well, which is covered on a US based website https://www.amyloidosis.org/facts .

Diagnosing amyloidosis can be challenging, and early detection is crucial for effective treatment. Symptoms can vary depending on the affected organs, but common signs include fatigue, weight loss, enlarged tongue, heart problems, kidney dysfunction, and nerve damage. Diagnostic tests may involve biopsies, imaging studies, blood tests, and genetic analysis, depending on the suspected type of amyloidosis.

Treatment options depend on the specific type and severity of amyloidosis. They may include chemotherapy, stem cell transplantation, medications to control underlying conditions, and supportive therapies to manage organ complications. Clinical trials and ongoing research aim to develop novel treatments and improve patient outcomes.

Despite being classified as a rare disease by the U.S. Office of Rare Diseases, experts believe that some types of amyloidosis may be underdiagnosed rather than truly rare. We believe the same is true here in New Zealand. Increasing awareness among healthcare professionals and the general public is crucial for early diagnosis and proper management of amyloidosis.

Our goal is to raise awareness, provide reliable information, and support individuals and their loved ones affected by amyloidosis.

On this website, you will find detailed information about AL amyloidosis. For ATTR we recommend the resources provided by the New Zealand Amyloidosis Patient Association (NZAPA).

Please navigate through the different pages on this website. It will evolve over time, so please revisit the site from time to time.

Please send your feedback to the email address provided..

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Amyloidosis is a group of diseases characterized by the production of abnormal proteins called amyloid. These proteins can accumulate and deposit in various organs, tissues, nerves, and other parts of the body, leading to impaired function and organ damage. It is important to note that there are

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Nothing can prepare you for Amy's story.

The years of suffering, the public humiliation, the trauma of watching her mother die.

On the day we meet Amy, she is thin. Desperately thin. Her 19-year-old son, Haze, the young man she is so enormously proud of, stands by her side. He is her protector, a young source of strength for his mother.

"He has a big heart and such a good soul," Amy says.

Amy tells me she wasn't always this thin. She has lost close to 40kgs in a matter of months and if she doesn't put weight on soon, she'll need a feeding tube.

She shudders, and then shakes her head.

"I don't want to be fed through my nose. I need to put on at least 5kgs. I'm trying."

The origins of Amy's illness stretch back more than a century. For as long as she can remember, there was talk of a Portuguese sailor who met one of Amy's British ancestors in Europe and fathered a child. The sailor was a carrier of a rare, mutant gene that medical scientists, a century later, would trace back to three localised clusters in Portugal, Ireland, and Japan.

The rogue Portuguese gene arrived in New Zealand in the DNA of one of Amy's long forgotten ancestors, and ever since it has continued its destructive march through every generation of her family.

It was later given a medical name, 'Hereditary TTR Amyloidosis' but only after it had slowly developed in her maternal grandmother, mother, two aunts and an uncle. It killed them all.

Carriers of the gene don't automatically develop the disease, but when Amy was 29 years old, she realised it was no longer lying dormant in her DNA.

"I felt a tingling in my hands and that's the first sign. I knew what was coming."

The amyloidosis is now ravaging Amy's body and challenging her will to live every day. She is now 39 years old, and surviving as best she can in a Kāinga Ora house in a quiet suburb in Porirua. New medicines have been developed to treat TTR Amyloidosis, but they are not publicly funded in New Zealand. And tragically for Amy, once this disease starts to develop, no treatment or medicine can reverse the cruel damage it inflicts.

"I don't care about me," she says. "It's too late for me now. Please, just help my son. Help Haze. That's all I want."

Amy (Te Whānau-ā-Apanui/Ngāti Porou) has raised Haze on her own, loving and caring for the boy she calls 'Bubba' for the last 19 years.

She has never spoken publicly about her disease, but that was before a recent test confirmed that Haze is a carrier too. If he develops the disease, Amy is desperate to make sure it stops with him, and her only child gets access to the right medicines to treat it.

"He was the best little kid. He never gave me any grief. He backchatted a couple of times when he was 13, but that was about it. He's always been a good boy."

Haze drops his head, shy and a little embarrassed at his mother's praise.

Then he looks at his mum. "We've been through a lot, eh Mum? We don't really talk about it much, but we've been through a lot together."

"Yep, we have Bub," she says.

TTR Amyloidosis causes an abnormal build-up of the amyloid protein in the body's organs and tissues. It strikes the nervous system first and then starts to impact the heart, eyes, and the gastrointestinal tract.

The primary form of treatment in New Zealand is a liver transplant to try and stop the rogue production of amyloid, but despite a successful transplant, amyloid continues to build-up in Amy's system.

She has a pacemaker too, but she says her heart is enlarged and the amyloid deposits cause it to 'stiffen'.

Her plummeting blood pressure is a constant battle. She blacks out, collapsing heavily on the floor, six or seven times a day.

"I've got a removable showerhead now, so I don't have to tilt my head back when I wash my hair. That's stopped me fainting in the shower, but I can't hang out the washing. I just black out and hit the floor."

Haze says when his mother faints at home, he hears a loud bang when she hits the floor or falls onto furniture. In the seconds that follow, he doesn't know what he's going to find.

Amy is suddenly tearful and drops her gaze to the floor.

"It's horrible for him. I don't know what's going on, and when I come around I either scream at him to get out, or yell at him to leave me alone. I don't know what I'm doing. And I've got no control over my bowel, so I soil myself when I faint. I'm his Mum....I don't want him to see me like that."

Amy says she tries to get to her room where she can sleep for an hour or two.

"Haze normally just waits in the house. He waits for me to wake up...."

Her son keeps his head down and tries to speak but he can't. There are tears in his eyes too. He's 19 years old, but he's already lived a lifetime. He had a 50-50 change of inheriting the mutant gene and the odds went against him. The disease is dormant for now, but the photos on the wall remind Haze every day that it has ravaged generations of his family.

There is no treatment available to reverse the damage caused by Hereditary TTR Amyloidosis but new medicines have been developed to specifically treat this disease, and prevent or delay its progression. New Zealand doesn't publicly fund these medicines.

In Auckland, some patients are involved in two international clinical trials for amyloidosis, the most exciting of which uses a CRISPR-Cas9 gene editing treatment, a one-off therapy that disables the mutant gene that causes this wretched disease.

It is, unquestionably, revolutionary, and as one biomedical engineer said, gene editing is ushering in "a whole new era of medicine".

However, our Ministry of Health has provided no thinking in this space, nor given any future consideration as to how our health system will respond to some of these - and many other - rapid developments in the treatment of disease.

In the past decade, successive New Zealand governments have, at best, taken a position of 'slightly interested bystander'.

One of the drugs available to treat Hereditary TTR Amyloidosis is the Pfizer-developed, Tafamadis. If the disease begins to manifest in Haze, Tafamadis would likely be the drug his specialist would consider. However, despite being recommended for funding here, negotiations between Pharmac and Pfizer have stalled. And, at $160,000 + GST a year, it's out of reach for the vast majority of New Zealanders. Not least for a young Māori boy from Porirua.

Amy's condition is worsening and there is little support for her as she battles this rare disease.

If she has a specialist appointment, she won't eat for 36 hours beforehand because her gastrointestinal tract is failing. She is terrified of losing control of her bowel and defecating in public and she can't afford adult diapers.

"$20 for four diapers is...it's just too expensive."

This mutant gene is robbing Amy of so much, but not least her dignity.

In public, few people understand that she is ill. She has a disabled badge on the windscreen of her car, but she has been spat on for parking in a disabled park.

"People think I'm on drugs. I'm thin, I can't walk very fast and I'm Māori with tattoos, but not everyone who is sick needs a wheelchair."

On another day, when she tried to park in a disabled park, a woman stood in front of her, with her hands on her hips.

"There is no point trying to explain my disease. They won't understand it."

Suddenly a smile creeps across Amy's face.

"I just sat in my car until she left but I was thinking 'come on, Karen....move along'."

Haze, who for so many years depended on his mother for love and care, is now the nurturer.

He has picked up some work with a construction company which he hopes will become fulltime, and he is supported by a group of kind and compassionate young friends.

"They're awesome. They know. They understand. They're always there for me," he says.

Amy is grateful that Haze has a strong group of friends but when he isn't in the room, she tells me that she doesn't want Haze to see what she has seen. She was 16 when her mother died, and she is still haunted by her death.

"The ambulance men tried to bring her back to life. She was lying on the bed with her eyes open...I wish I had never seen her like that. It was 1998, and I still can't get that image out of my head."

Haze is heading outside into a cold Porirua southerly.

"Bubba, it's cold outside. Get a coat."

I notice the tattoo on Haze's arm. It's the same as his mother's.

"The world is yours," it says.

And it can be, if we agree to start working on a solution to New Zealand's unfunded medicines crisis.

Show your support.

Share Amy's story.

New Zealand needs access now to modern medicines to treat Amyloidosis.

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Nothing can prepare you for Amy's story. The years of suffering, the public humiliation, the trauma of watching her mother die. On the day we meet Amy, she [...]

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Preimplantation genetic testing for aneuploidy (PGT-A)

PGT-A is an analysis of embryo cells to determine if there is the normal amount of chromosomes. An unequal division of either sperm or egg cells can result in an embryo having too few or too many chromosomes.

Most people have 46 chromosomes because they inherit 23 chromosomes from each parent. If an embryo or a cell is missing a chromosome or has an extra one, it is called aneuploidy. Monosomy is a missing chromosome and trisomy is an extra chromosome.

A child can only survive one type of monosomy, Turner syndrome, which is the absence of one of the X chromosomes. Trisomy of chromosome pairs can sometimes result in live birth, Down syndrome, also called trisomy 21 (an extra chromosome in normal pair # 21), Turner syndrome (trisomy 18) and Patau syndrome (trisomy 13). Down syndrome affects 1 in 700 babies, according to the Centers for Disease Control and Prevention.

Aneuploidy is one of the greatest causes of failed implantation for pregnancy and miscarriage, as well as a major cause of birth defects in children.

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Embryo Preimplantation Genetic Testing (PGT) | Fertility Center

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Preimplantation genetic testing for aneuploidy (PGT-A) PGT-A is an analysis of embryo cells to determine if there is the normal amount of chromosomes. An unequal division of either sperm or egg cells can result in an embryo having too few or too many chromosomes.

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Preimplantation genetic testing (PGT) is a cutting-edge procedure used to identify genetic abnormalities in embryos created with in vitro fertilization (IVF). The goal of PGT is to allow your physician to select embryos predicted to be free of a specific genetic condition or chromosome abnormalities for transfer. This test provides patients with an opportunity to reduce the chance for a genetic disease in their future children prior to pregnancy.

How it works

If you are interested in PGT, the first step is to notify your physician. Determining whether you should have PGT performed for your embryos is a complex decision so careful discussion with your physician is important. We also strongly recommend genetic counseling for all our patients who are considering PGT. Like all genetic tests, PGT has important benefits and limitations of which patients should be aware. The decision to have PGT needs to be made well in advance of your IVF cycle.

If PGT is part of your plan, you will need to undergo an IVF cycle to retrieve eggs and create embryos. The embryos are monitored in our laboratory until day 5 or 6 after fertilization when they are referred to as blastocysts. At that point, a small number of cells are removed from each embryo and shipped to an outside laboratory for PGT. The Fertility & Reproductive Center partners with several reference laboratories that perform PGT including Natera and Cooper Genomics.

While PGT is being performed on the DNA from the cells that were removed, the embryos are cryopreserved (frozen) and stored in our laboratory. Once the PGT results are available, typically in 1-2 weeks after the biopsy, you will be notified of the results. If you have embryos that are appropriate for transfer, you will schedule a frozen embryo transfer with your physician.

Biopsy being performed on an embryo at the blastocyst stage. Typically, 5-10 cells are removed for genetic testing.

Testing to avoid inheritance of a single gene disorder (PGT-M)

Some patients seek IVF with PGT to reduce the chance of a specific genetic condition occurring in their children. This type of PGT is called preimplantation genetic testing for monogenic disorders (PGT-M). In some cases, the patient themselves may have a genetic condition such as neurofibromatosis type 1 or Marfan syndrome that could be passed on to their children. In other cases, both members of a couple may be carriers for a recessive genetic condition such as cystic fibrosis or sickle cell anemia, or the individual using their eggs may be a carrier for an X-linked condition such as Fragile X syndrome. HLA matching is also available for families with certain blood disorders who may wish for an embryo that could be an HLA match for a sibling or other family member.

Our physicians and genetic counselor work closely with such patients and an outside laboratory to design personalized testing for their embryos. The goal of PGT-M is to predict which embryos are free of the genetic condition for which they are at risk, allowing the physician to select those embryos for transfer. PGT-M can greatly reduce the risk of having an affected child prior to pregnancy.

Screening for chromosome abnormalities (PGT-A and PGT-SR)

PGT can also evaluate an embryo's chromosomes, the structures in every cell that contain our genes. It is not uncommon for embryos have random chromosome abnormalities such as missing or extra chromosomes, also known as aneuploidy. Embryos with aneuploidy are more likely to result in a miscarriage or failed IVF cycle. Less commonly, aneuploidy may result in the birth of a child with a chromosome condition such as Down syndrome (trisomy 21).

In a typical IVF cycle, embryos are chosen for transfer based on their appearance under the microscope, which is referred to as their grade. The embryo's appearance can give us some indication of its potential to result in a successful pregnancy, but it is not perfect. Preimplantation genetic testing for aneuploidy (PGT-A) provides additional information about the reproductive potential of the embryos and may help select the best embryo for transfer. PGT-A is most often considered for patient who have had recurrent pregnancy losses (miscarriages), multiple unexplained failed IVF cycles, a prior pregnancy or child with certain chromosome abnormalities, or based on the age of the individual providing their eggs. While PGT-A can be performed for any IVF cycle, there are both benefits and limitations that should be carefully discussed with your physician to determine if PGT-A is right for you.

One final type of PGT called PGT-SR is for structural rearrangements. This type of PGT is performed when a patient or their partner has a rearrangement of their own chromosomes such as a translocation or inversion. A person with a structural rearrangement of their own chromosomes is at increased risk to produce embryos with missing or extra pieces of chromosomes. Embryos with missing or extra pieces of chromosomes are more likely to result in a miscarriage or a child with serious health issues. PGT-SR can help to reduce those risks.

Questions?

If you would like additional information about PGT, you can find the answers to many frequently asked questions here.

If you have additional questions or feel PGT may be right for you, please contact us at 314.286.2497

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Preimplantation genetic testing (PGT) is a cutting-edge procedure used to identify genetic abnormalities in embryos created with in vitro fertilization (IVF). The goal of PGT is to allow your physician to select embryos predicted to be free of a specific genetic condition or chromosome abnormalities for transfer.

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PGT-SR: A Comprehensive Overview and a Requiem for the Interchromosomal Effect

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Preimplantation genetic testing for structural rearrangements (PGT-SR) was one of the first applications of PGT, with initial cases being worked up in the Delhanty lab. It is the least well-known of the various forms of PGT but nonetheless provides effective treatment for many carrier couples. Structural chromosomal rearrangements (SRs) lead to infertility, repeated implantation failure, pregnancy loss, and congenitally affected children, despite the balanced parent carrier having no obvious phenotype. A high risk of generating chromosomally unbalanced gametes and embryos is the rationale for PGT-SR, aiming to select for those that are chromosomally normal, or at least balanced like the carrier parent. PGT-SR largely uses the same technology as PGT-A, i.e., initially FISH, superseded by array CGH, SNP arrays, Karyomapping, and, most recently, next-generation sequencing (NGS). Trophectoderm biopsy is now the most widely used sampling approach of all PGT variants, though there are prospects for non-invasive methods. In PGT-SR, the most significant limiting factor is the availability of normal or balanced embryo(s) for transfer. Factors directly affecting this are rearrangement type, chromosomes involved, and sex of the carrier parent. De novo aneuploidy, especially for older mothers, is a common limiting factor. PGT-SR studies provide a wealth of information, much of which can be useful to genetic counselors and the patients they treat. It is applicable in the fundamental study of basic chromosomal biology, in particular the purported existence of an interchromosomal effect (ICE). An ICE means essentially that the existence of one chromosomal defect (e.g., brought about by malsegregation of translocation chromosomes) can perpetuate the existence of others (e.g., de novo aneuploidy). Recent large cohort studies of PGT-SR patients seem, however, to have laid this notion to rest, at least for human embryonic development. Unless new evidence comes to light, this comprehensive review should serve as a requiem.

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The IP address used for your Internet connection is part of a subnet that has been blocked from access to PubMed Central. Addresses across the entire subnet were used to download content in bulk, in violation of the terms of the PMC Copyright Notice.

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Fertility technology you may to consider with IVF

Fertility technology you may to consider want with IVF:
Embryo Freezing
Often more than two or three embryos are produced in an IVF cycle. Good quality 'spare' embryos can be frozen, and later thawed to give another chance of pregnancy. Freezing and thawing have to be done under special conditions: >90% of embryos can survive the procedure. When embryos are to be thawed the woman's menstrual cycle is monitored with blood tests to make sure the embryos are replaced at the right time of the menstrual cycle.
Thaw cycle of IVF
When there have been embryos frozen following in vitro fertilisation, they can be thawed and transferred into the uterus at a later stage. This usually entails several bloods tests to time ovulation during a natural cycle. In some cases it is necessary to control a cycle using drugs to prepare the uterus for implantation. Embryos are transferred into the uterus at the appropriate time, as they are in an IVF cycle.

ICSI is a variation of IVF. Instead of the sperm and eggs being mixed together, a single sperm is injected into each mature egg. ICSI may be used, for example, when sperm quality is too poor for conventional IVF to work. ICSI allows almost any man with sperm, either in his semen or in his testes, to try IVF.

Photographs embryos every 10 minutes to capture developmental milestones in the embryo's life that are missed when embryos are only inspected once a day.

Benefits of TiMI:

Picks up unusual and detrimental events that escape a daily snap-shot inspection.

Identifies 10-15% of embryos with very low potential.

Several studies show that TiMI increases the chance of pregnancy using the first embryo transferred by up to 10%.

Embryos are not removed from their incubator, therefore they develop in an uninterrupted environment.
Who can benefit from TiMI?

People who expect to have several good quality embryos to choose from.

People who have had low quality embryos previously and want more information about their embryo development.

Embryo selection with TiMI does not increase the overall chance of a baby from all the embryos available, but it can reduce the time to pregnancy by giving a better choice of which embryo to use first.

Pre-implantation genetic testing can identify genetic abnormalities in embryos at the blastocyst stage (a blastocyst is an embryo that has been developed in the laboratory for five or six days after fertilisation). This allows us to select the 'best' embryos which are most likely to be free of genetic conditions and chromosomal abnormalities for transfer to the uterus.

Because many blastocysts have the wrong number of chromosomes (a condition known as aneuploidy), PGT is a powerful tool for selecting a good embryo for transfer. Aneuploidy can lead to miscarriages, birth defects and other complications.
Types of PGT:
PGT-M: Pre-implantation genetic testing for monogenic/single gene disorders

This is for those who may have a known genetic condition that they do not want to pass on to their children. PGT means that couples can have a child that is fully theirs genetically, without the child inheriting the particular genetic disorder.

Examples of conditions that may be excluded by this test are: neurofibromatosis type 1; Marfan syndrome; cystic fibrosis; Huntington's; muscular dystrophy.

How can TiMI & PGT work together?
Preimplantation genetic testing (PGT) is the strongest embryo selection tool we have since we know that many normal appearing embryos have the wrong number of chromosomes (aneuploidy).

The challenge with PGT is that it requires blastocysts on day 5 or 6 for testing - probably 3 or more. TiMI complements PGT because it provides the best undisturbed environment we have available in order to get as many blastocysts as possible. While the extra information we gain from watching the embryos with TiMI is helpful when not using PGS, it does not add to the selection process when PGS is used.

The challenge most patients face is knowing how many embryos they'll have to work with. If the number is few, then TiMI is a safe way to increase your chances. If there are 3 or more blastocysts, then PGS alone or in combination with TiMI may be the better option.

PGT can be used by people who have a chance of passing serious genetic disorders on to their children, such as:

Cystic Fibrosis, Huntington's, Beta-thalessemia, Fragile X and Spinal Muscular Atrophy. These are caused by a change in single gene.

People who have a high chance of pregnancy loss or a lower chance of pregnancy because of translocations between chromosomes.

Useful to know:
Ethics Committee approval is required to carry out PGD for any other reason, (such as 'saviour siblings'). PGD cannot be used for gender selection in New Zealand.

Genetic carrier screening gives individuals and couple's information about their risk of having a child with a genetic condition.

We now offer the option to do genetic testing through Invitae, either for your own information or to check compatibility with your partner or potential donor.

Genetic screening identifies whether you are a carrier of a range of inherited conditions. Most people are carriers of one or more genetic conditions, even though no one in their family has the condition. Being a carrier is important if the person using the sperm is also a carrier of the same condition.
Invitae's carrier screening includes:

Severe and prevalent disorders seen across all ethnicities

Enhanced SMA testing to help identify silent carriers

Comprehensive Fragile X analysis, including AGG interruptions

Full gene sequencing with deletion and duplication analysis leading to a 99% detection rate for most genes

Actionable results; no reporting of variants of unknown significance

This is a method whereby the endometrium may be checked to determine whether the date of embryo transfer may need to be adjusted to match the woman's own window for implantation.

For most women the time of endometrial receptivity for an embryo to implant is accurately predicted by the number of days after ovulation, but for some women the 'window for implantation' occurs later or earlier. The ERA is a method whereby the endometrium may be checked to determine whether the date of embryo transfer may need to be adjusted to match the woman's own window for implantation.
How it works:

The woman goes through a mock frozen embryo transfer cycle and on the day of embryo transfer instead of replacing an embryo, a Pipelle biopsy is done. This is where a small sample of the lining or endometrium is taken and then couriered to the ERA lab. (For NZ the nearest lab is Japan.)

The ERA lab then checks the expression of 238 genes in the biopsy sample and sends a report indicating a receptive endometrium or a pre-receptive or post-receptive endometrium.

The timing of the embryo transfer is then adjusted accordingly.
Who should do this test?
The current recommendations are that the ERA may be useful in women with recurrent implantation failure. There is debate about what the definition of implantation failure is - we consider ERA if a pregnancy has not resulted after the transfer of 2 to 3 good quality embryos into a normal uterus, in which the expectation of greater than 80% would have become pregnant.

This is an option in IVF or ICSI. A small hole is made in the soft shell of the embryo before it is replaced in the uterus.

It is possible to detect/screen for fetal abnormalities using non-invasive prenatal testing 'NIPT', Chorionic Villus Sampling 'CVS' or Amniocentesis.

NIPT (non-invasive prenatal testing) - available in our Auckland clinic only - is a blood test performed any time from 10 weeks of pregnancy. The results are available within 5-10 days. NIPT uses cell-free fetal DNA (cfDNA) found in maternal blood to identify the most common chromosome conditions seen in newborns, Trisomy 21 (Down Syndrome), Trisomy 18, Trisomy 13 and sex chromosome abnormalities including Turner syndrome, Klinefelter syndrome , Triple X and XYY syndrome.

CVS Chorionic Villus Sampling or Amniocentesis can also be used to confirm a diagnosis in those with a higher risk from the screening result. Screening is free for CVS or Amniocentesis for those at higher risk.

Please ask your Obstetrician, Genetic Counsellor or Lead Maternity Carer for more information.

Several studies have shown that a small amount of the hormone human Chorionic Gonadotrophin (hCG) infused into the uterus shortly before embryo transfer may increase the chance of an embryo implanting.

The chance of an embryo implanting depends on the quality of the embryo and the receptivity of the uterus. Little is known about how to influence the receptivity of the uterus, but there have been several studies looking at the infusion of a small amount of the hormone hCG before embryo transfer.

A recent meta-analysis looked at the results from 15 randomised control studies that included over 2500 patients. The conclusion was that on average infusion of hCG increased the chance of a live birth after embryo transfer. It also appeared to lower the chance of miscarriage. hCG infusion seemed to be more effective for the transfer of fresh embryos than frozen embryos. An infusion within 15 minutes of embryo transfer seemed to be more effective than an infusion done earlier, and a dose of around 500iu seemed to be better than higher doses.
Overall, the evidence for a potentially beneficial effect of hCG infusion is quite high, so this is one of the add-ons which we classify as of positive benefit.

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Fertility technology you may to consider want with IVF: Embryo Freezing Often more than two or three embryos are produced in an IVF cycle. Good quality 'spare' embryos can be frozen, and later thawed to give another chance of pregnancy. Freezing and thawing have to be done under special conditions: >90% of embryos can survive the procedure.

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IVF, Fertility Treatment, Fertility Specialists, Repromed

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Genetic screening | Fertility Specialists | Repromed

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A range of genetic testing options can be carried out at different stages of your fertility journey, starting even before you become pregnant.

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There's limited research into whether PGT-A improves success rates.

It can be expensive and is not available on the NHS.

Your doctor should explain how PGT-A might help you.

What is PGT-A?

PGT-A (previously known as preimplantation genetic screening or PGS) is a treatment add-on. It involves checking embryos created by in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) for abnormalities in the number of chromosomes. PGT-A doesn't check for specific genetic diagnoses.

During PGT-A, a single cell or a small number of cells is removed from the embryo. The DNA of these cells is then tested to see whether they have any chromosomal abnormalities. Only embryos without chromosomal abnormalities are placed back in the womb.

are more likely to have embryos with chromosomal problems

Who might be recommended to have PGT-A?

Older women are more likely to have eggs with the wrong number of chromosomes, so traditionally it's been offered to women over 37. Abnormal chromosomes are thought to be the main reason why older women have difficulties conceiving and are more likely to have a miscarriage or a baby with Down's Syndrome.

Your doctor may also recommend PGT-A if you have a family history of chromosome problems, if your sperm is at risk of carrying abnormal chromosomes, or if you've had several miscarriages or failed IVF attempts without explanation.

However, there is limited evidence to show that PGT-A benefits these groups (see more below). It's also not a guaranteed way to prevent miscarriage or to have a baby.

What causes abnormal chromosomes?

When the cells of eggs, sperm and embryos divide this can lead to too many, or too few, chromosomes, or with missing or added pieces of DNA. Abnormal chromosomes happen very frequently during the normal production of eggs, sperm and during embryo development.

Does PGT-A work?

PGT-A is traditionally offered to women over 37 with a history of miscarriage or failed IVF cycles or people with a family history of chromosome problems. However, to date there is little evidence showing it improves the chances of having a baby for most fertility patients and therefore it is considered a treatment add-on.
Find out more about PGT-A on our treatment add-ons page
Until larger trials have been run and we have more evidence, there's no guarantee that PGT-A can improve your chances of a successful pregnancy.

How safe is it for the embryos?

PGT-A involves taking a biopsy, to remove a cell or number of cells, from an embryo which can sometimes cause damage to the embryo and prevent it from developing once it has been transferred into the womb.

Are there any other risks?

PGT-A carries the same risks as IVF - find out more about the risks of fertility treatment.

However, PGT-A also has some treatment-specific risks. This includes the possibility of a misdiagnosis (eg due to mosaicism described above). There is also the risk that, if all the embryos are found to have abnormal chromosomes, there won't be any embryos to put back in the womb. This is especially likely for older women.

Can I have PGT-A on the NHS?

No, PGT-A is not currently funded by the NHS. PGT-A can be expensive so you should talk to your clinic about likely costs and make sure your clinician clearly explains why PGT-A might be appropriate for you.

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Frequently asked questions about Pre-implantation genetic testing for aneuploidy (PGT-A)

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PGT-A (previously known as preimplantation genetic screening or PGS) is a treatment add-on.

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PCRM: Pacific Centre for Reproductive Medicine

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What is Preimplantation Genetic Testing? - PCRM Fertility Clinic

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Abstract

The past several decades have seen tremendous advances in the field of medical genetics. The application of genetic technologies to the field of reproductive medicine has ushered in a new era of medicine that is likely to greatly expand in the coming years. Concurrent with an in vitro fertilization (IVF) cycle, it is now possible to obtain a cellular biopsy from a developing embryo and genetically evaluate this sample with increasing sophistication and detail. Preimplantation genetic screening (PGS) is the practice of determining the presence of aneuploidy (either too many or too few chromosomes) in a developing embryo. However, how and in whom PGS should be offered is a topic of much debate.

Keywords: preimplantation genetic screening, PGS, embryo, recurrent pregnancy loss, microarray, FISH, PGD

Introduction

Over the past several decades the world has witnessed incredible advances in many medical fields. No field has seen more dramatic change, however, than the field of genetics. The mammoth explosion of technologies surrounding genetics is staggering. Sequencing the human genome was only a short time ago considered the "Mount Everest" of medical achievement. Today, sequencing an entire individual's genome is widely available and can be performed in a day (rather than years) for thousands (rather than millions) of dollars.1 In a similar manner, technology surrounding preimplantation genetic screening (PGS) has advanced greatly in recent years. In the following review we will outline PGS, current applications for this technology, and the limitations of the procedure.

An in vitro fertilization (IVF) cycle consists of administrating injectable gonadotropins to women and induce controlled ovarian hyperstimulation in which more than the usual number of ovarian follicles are recruited and matured.2,3 The oocytes within these follicles are then surgically harvested and inseminated with sperm. Typically, resultant embryos are then grown in vitro until either 3 or 5 days of development, at which time the 1 or 2 "best" embryos are placed into the uterus and the remaining embryos are cryopreserved. Determining which embryos are the "best" has been a subject of much debate since the birth of the technology in the late 1970's. Traditionally the use of morphology, the visual appearance of embryos, has been the principal modality of choosing optimal embryos for uterine transfer.4 However, the implantation rate per transferred embryo in most clinics rarely exceeds 40%.5 Therefore, many investigators have for some time been searching for other diagnostic methods capable of more accurately determining embryo quality than morphology alone. This effort has produced several promising technologies including metabolomics, real-time videography, and PGS.

The Evolution of PGS

PGS is the practice of taking a biopsy either from the polar body of a mature oocyte or from cells taken from developing embryos and genetically analyzing the composition of these cells. Results of this genetic analysis direct the embryologist in choosing embryos for uterine transfer. Because PGS can only be performed using cells obtained at embryo biopsy, this technology is only possible in conjunction with an in vitro fertilization (IVF) cycle. PGS is the practice of evaluating embryos for chromosomal aneuploidy, the presence of either too many or too few chromosomes, in chromosomally normal parents. In contrast, preimplantation genetics diagnosis (PGD) is the practice of evaluating embryos for specific genetic abnormalities, such as sickle cell disease or cystic fibrosis, where carrier status has been documented in each of the parents.

Certain patient populations, including couples with advanced maternal age, recurrent miscarriage, repeated implantation failure, and severe male factor, are thought to have a predisposition for producing aneuploid embryos.3,6,7 Many have suggested that these patient populations may be benefit from PGS.3,7 However, the indications for using PGS in many centers is constantly expanding.3 As reported by the European Society of Human Reproduction and Embryology (ESHRE), over the past 10 years, 61% of all preimplantation genetic testing cycles were performed for PGS.8 This paper will focus exclusively on the clinical applications associated with PGS rather than PGD.

PGS, unlike PGD, has been and continues to be a controversial technology. Recent studies indicate that greater than 60%-90% of all first trimester miscarriages may be the result of chromosomal aneuploidy.3 Because so many early miscarriages are due to aneuploidy, PGS seems to be a reasonable intervention to improve the efficiency with which euploid (chromosomally normal) embryos are selected for uterine transfer in IVF cycles. Classic studies have reported that miscarriages that are caused by aneuploidy are disproportionally concentrated on select chromosomes.9,10 These data are based on karyotype analysis of failed pregnancies that developed far enough along to have tissue available for genetic analysis.9,10 Consequently, clinics performing PGS in the early days of the technology focused on detecting aneuploidy on only select chromosomes using fluorescence in situ hybridization (FISH), which typically evaluates between 5-14 chromosome pairs rather than all 23 chromosome pairs.11,12 Traditionally, PGS biopsy was exclusively performed at approximately 3 days of embryonic development following fertilization.11,12 Initial data using PGS in the context of cleavage stage biopsy with FISH showed promising results and generated much excitement for this new technology.3,13⁻15 Unfortunately the results from this approach failed to result in improvements in clinical pregnancy rates and this lack of efficacy was widely referenced following a landmark paper by Mastenbroek in the New England Journal of Medicine.16 Subsequently, similar papers cast further doubt on the benefits of PGS and position statements from major medical societies formally discouraged its use.17⁻19

Further research, however, elucidated several biologic limitations that could explain the prior shortcomings of clinically applied PGS. The practice of polar body biopsy to determine the genetic composition of a fertilized oocyte is a commonly utilized modality for performing preimplantation genetic testing.3,8,20 A critical component of oocyte development is meiotic division in which a haploid set of unused maternal DNA is marginalized into what is termed a polar body.3,8 Genetic evaluation of this polar body was initially quite popular, as this process obtained a diagnosis without disturbing the developing embryo and could be performed prior to fertilization.3 However, this approach is incapable of detecting paternally derived genetic errors, or any errors introduced after or during fertilization. Due to these limitations, polar body biopsy is now principally performed in countries where strict legislation limits the practice of embryo biopsy.3,21

However, PGS using biopsied cells from developing embryos also presents challenges. Studies have repeatedly documented that embryos at day 3 of development have high levels of mosaicism.22,23 Mosaicism is a condition in which a single developing embryo is comprised of more than one distinct genetic cell line. In other words, mosaic embryos may have euploid (normal) and aneuploid (abnormal) cell lines within a single embryo. Studies evaluating this phenomenon have concluded the majority of all embryos may be mosaic at day 3 of development.22⁻24 Consequently, a biopsy performed at day 3 of development may produce a result that is not representative of the entire embryo.3 Mosaicism has been shown to also exist at day 5 of embryo development.25 However, recent data suggests that mosaicism may be much reduced by day 5 of development.3,26

Another limitation of traditionally performed PGS was the use of FISH for determination of chromosomal abnormalities. FISH typically evaluates between 5-14 rather than all 23 chromosome pairs.27 Recent studies have indicated that embryonic aneuploidy occurs in clinically significant amounts in all 23 chromosome pairs.28 Therefore, FISH is incapable of diagnosing many of the chromosomal abnormalities commonly found in developing embryos.

Realization of these two principal limitations has led many genetic laboratories to offer PGS using technologies evaluating the chromosomal status of all 23 chromosome pairs using an embryonic biopsy performed at the blastocyst stage, typically reached by day 5 or 6 of development. The clinical pregnancy rates using this approach have been reported to be markedly superior to the traditional approach of performing PGS.29,30 For example, a recent study evaluating more than 4,500 embryos using 23 chromosome pair determinations found clinical pregnancy rates in women suffering from recurrent pregnancy loss (RPL) to be significantly improved above similar studies using FISH PGS.29 Additionally, pregnancy rates were further improved when 23 chromosome evaluation PGS was performed on blastocyst stage embryos (day 5/6 of development) as compared to when the biopsy was performed on embryos at day 3 of development. 29,31,32 Similar results have been reported consistently by many clinics in the United States and around the world.31,32 This has generated a renewed interest in PGS, although it still remains to be determined whether PGS is an efficacious technology, and which patient populations are best served by PGS.

Evaluation of all 23 chromosomes in the context of PGS possesses inherent complexities that potentially may compromise the integrity of data if not properly performed. There are multiple approaches that are used to perform 23 chromosome pair evaluation. The two modalities that are most commonly used today utilize microarray technology, either by using single nucleotide polymorphism (SNP) or comparative genomic hybridization (CGH) technology.3 Both of these technologies rely on obtaining embryonic DNA, fragmenting and then amplifying this DNA, and evaluating this amplified product using microarrays. This amplification process is a potential source of error, as failure to amplify the entire embryonic DNA product could produce a false result. Additionally, because the DNA product being initially amplified is taken from only 1 to several cells, any external DNA contamination can produce a spurious result.

SNP arrays directly evaluate ploidy status using a dense array of approximately 300,000 genetic markers.3 CGH arrays, in contrast, evaluate far fewer genetic markers and compare this result to a known normal DNA sample.3 Each of these microarray platforms have advantages and disadvantages. A purported advantage of SNP arrays is their ability to detect relatively small genetic duplications or deletions, though the value of this information is, at the present time, generally unclear. An advantage of CGH arrays is that they may be performed in 12-16 hours as opposed to several days for most SNP arrays.

Evidence for the Clinical Application of PGS

Studies from centers using PGS on day 5 and 6 blastocysts show promising results with transferred embryos generating pregnancy rates greater than 75%.29,31,32 However, a central criticism of the widespread use of PGS is a lack of randomized controlled trials that conclusively show the procedure to be beneficial. To our knowledge, at the time of writing this article, there is only one randomized trial to show a pregnancy benefit using PGS versus IVF using morphology alone in the setting of exclusive single embryo uterine transfer.33 This study was relatively small and had several significant limitations. Therefore, further larger and more rigorously studies are required before PGS will be more broadly accepted.3 Several large randomized controlled clinical trials are currently underway that will hopefully provide such data in the near future.

Despite the lack of support from professional societies and lack of large randomized controlled trials definitively demonstrating the benefits of the technology, PGS comprises the majority of all preimplantation genetic testing internationally and is being increasingly utilized.8 However, the patient population in which PGS may be appropriate is unclear at the present time.3 Many PGS clinics have traditionally recommended PGS for couples with risk factors believed to be associated with embryonic aneuploidy such as unexplained RPL, severe male factor, and advanced maternal age. However, in recent years many clinics have liberally expanded the use of PGS to many women without such risk factors. In fact, some clinics broadly recommend PGS to virtually all IVF patients as a strategy to improve pregnancy rates in couples battling infertility. The debate surrounding the appropriate patient populations for PGS is currently in flux and will likely be a source of debate for years to come.

Limitations of PGS

Despite the positive data that is emerging within the field of PGS, there are tangible technical and biological limitations to the technology. The limitations of FISH PGS evaluation and the use of biopsy taken from day 3 embryos are significant and have been discussed previously. Additionally, technical limitations surrounding the use of both SNP and CGH arrays may produce spurious results if not properly executed.3 Furthermore, while automated results are generated, the raw data is also interpreted by a trained geneticist. Therefore, the interpretation of results may be subjectively interpreted and this leaves inherent room for human error in addition to inaccurate automated resulting.

Perhaps the most significant source of error from PGS using 23 chromosome pair evaluation and blastocyst biopsy (day 5/6 of development) is the presence of cellular discordance within the developing embryo. A blastocyst embryo is comprised of 2 components, an inner cell mass (ICM) and trophectoderm (TE).3 The ICM possesses cells that are destined to form fetal tissue and the TE possesses cells that will form the placenta. Blastocyst biopsy utilizes cells taken from the TE to minimize potential deleterious effects that may be caused from biopsy of the ICM, cells destined to form the fetus. Though there is clearly a high degree of correspondence between the genetic composition of the ICM and the TE, some data suggests that in up to 10% of developing blastocysts, there may be aneuploidy present in the TE but not ICM or vice versa.26 Therefore, TE biopsy taken at the blastocyst stage, from a biological standpoint, may not be universally predictive of the chromosomal status of the developing embryo even if no technical error exists in the performance of genetic analysis.3 Mosaicism may also exist within a given TE cell population. Array technology, however, is capable of detecting all but very low levels of mosaicism within TE samples analyzed provided.26,34 The above limitations of PGS demand that patients be adequately counseled on the risks and limitations of PGS, preferably with the aid of a specialized physician, geneticist, or genetic counselor. Furthermore, antenatal genetic testing is still recommended in all patients undergoing PGS.3

Conclusion

New technologies associated with PGS are producing data that suggest that the procedure could be a valuable adjunct to assisted reproductive technologies in the future to enhance pregnancy success in many patients. Defining the exact benefit conferred by PGS and determining exactly which patient populations could be best served by PGS, however, is currently controversial. In the near future, several large and high quality studies will attempt to answer these questions. Despite the current lack of definitive data, PGS is increasingly being applied to patients with ever-expanding clinical indications. While there are few large randomized controlled studies defining the benefits of PGS, clinical data emerging from many PGS laboratories around the world is encouraging. Therefore, the judicious use of PGS seems reasonable at the present time, as the preponderance of available current evidence suggests that some couples may see a benefit from this technology. Only with time will the role of PGS be clearly defined.

Acknowledgments

We thank Dr. Jianchi Ding for help with the writing of this manuscript.

Footnotes

Contributors
PRB and WHK primarily searched the literature. RWK performed literature review. PRB wrote the first draft of the manuscript. RWK participated in writing and provided review assistance. WHK advised on the content of the manuscript regarding generalist physicians, assisted in the literature search, and contributed to the writing of the manuscript. PRB is guarantor.
Competing Interests
Author(s) disclose no potential conflicts of interest.
Disclosures and Ethics
As a requirement of publication author(s) have provided to the publisher signed confirmation of compliance with legal and ethical obligations including but not limited to the following: authorship and contributorship, conflicts of interest, privacy and confidentiality and (where applicable) protection of human and animal research subjects. The authors have read and confirmed their agreement with the ICMJE authorship and conflict of interest criteria. The authors have also confirmed that this article is unique and not under consideration or published in any other publication, and that they have permission from rights holders to reproduce any copyrighted material. Any disclosures are made in this section. The external blind peer reviewers report no conflicts of interest. Provenance: the authors were invited to submit this paper.
Funding
Author(s) disclose no funding sources.

References

9. Monni G, Ibba RM, Zoppi MA. Prenatal Genetic Diagnosis through Chorionic Villus Sampling. In: Milunsky A, Milunsky J, editors. Genetic Disorders and the Fetus. 6th ed. Oxford: Wiley-Blackwell; 2010. [ Google Scholar]

Articles from Clinical Medicine Insights. Reproductive Health are provided here courtesy of SAGE Publications

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Preimplantation Genetic Screening: A Practical Guide

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The past several decades have seen tremendous advances in the field of medical genetics. The application of genetic technologies to the field of reproductive medicine has ushered in a new era of medicine that is likely to greatly expand in the coming ...

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Genetic Screening and Genetic Testing Explained

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PGS, PGT-A, PGT-M, PGD… what does it all mean?

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Fertility Society of Australia and New Zealand

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Please note that in Australia and New Zealand, specific policies apply around the legality of available tests. In particular, when testing creates a situation where couples could apply sex selection when selecting an embryo, states have particular legislation in place.

In New Zealand, preimplantation genetic diagnosis is subject to different ethical views, and there are guidelines for fertility specialists.

Ask your fertility specialist which legislation applies to your local area before making decisions about your preimplantation genetic testing.

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Genetic testing (PGT) in Australia & NZ | Fertility Society

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Preimplantation Genetic Testing in AU and New Zealand. Screening cells from embryos for genetic diseases & chromosomal disorders: what can & cannot be tested?

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How in vitro fertilization (IVF) works - Nassim Assefi and Brian A. Levine

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Use this source above and the site below as sources of information to learn about the;

> Why

>How

>Processes

>Risks

>Key Biology concepts

https://www.mayoclinic.org/tests-procedures/in-vitro-fertilization/about/pac-20384716

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IVF: A Biological Milestone - Trio Sci Cymru

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What is IVF? I’m sure you all know the basics behind natural conception, once the sperm has fertilised the egg and embryo is formed. In natural pregnancy this embryo would then attach itself to the uterus wall, or womb and begin developing. However, if natural or unassisted conception is not possible, fertility treatment is an … IVF: A Biological Milestone Read More »

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How Does In Vitro Fertilization (IVF) Work? A Step-by-Step Explanation

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How CRISPR lets you edit DNA - Andrea M. Henle

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○ genome analysis and genomic selection methodologies ○ DNA sequencing ○ Digestion of DNA by restriction enzymes ○ Gel electrophoresis ○ PCR-based methodologies ○ Gene cloning and transformation/transfection techniques such as ligation and use of plasmids/tumour inducing plasmids or bacteriophage vectors, selection methodologies for the transformant ○ Guided nuclease-mediated gene targeting, such as clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) or TALEN.

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A British toddler has had her hearing restored after becoming the first person in the world to take part in a pioneering gene therapy trial, in a development that doctors say marks a new era in treating deafness.

Opal Sandy was born unable to hear anything due to auditory neuropathy, a condition that disrupts nerve impulses travelling from the inner ear to the brain and can be caused by a faulty gene.

But after receiving an infusion containing a working copy of the gene during groundbreaking surgery that took just 16 minutes, the 18-month-old can hear almost perfectly and enjoys playing with toy drums.

Her parents were left "gobsmacked" when they realised she could hear for the first time after the treatment. "I couldn't really believe it," Opal's mother, Jo Sandy, said. "It was ... bonkers."

The girl, from Oxfordshire, was treated at Addenbrooke's hospital, part of Cambridge university hospitals NHS foundation trust, which is running the Chord trial. More deaf children from the UK, Spain and the US are being recruited to the trial and will all be followed up for five years.

Prof Manohar Bance, an ear surgeon at the trust and chief investigator for the trial, said the initial results were "better than I hoped or expected" and could cure patients with this type of deafness.

"We have results from [Opal] which are very spectacular - so close to normal hearing restoration. So we do hope it could be a potential cure."

He added: "There's been so much work, decades of work ... to finally see something that actually worked in humans .... It was quite spectacular and a bit awe-inspiring really. It felt very special."

Auditory neuropathy can be caused by a fault in the OTOF gene, which makes a protein called otoferlin. This enables cells in the ear to communicate with the hearing nerve. To overcome the fault, the new therapy from biotech firm Regeneron sends a working copy of the gene to the ear.

A second child has also recently received the gene therapy treatment at Cambridge university hospitals, with positive results.

The overall Chord trial consists of three parts, with three deaf children including Opal receiving a low dose of gene therapy in one ear only.

A different set of three children will get a high dose on one side. Then, if that is shown to be safe, more children will receive a dose in both ears at the same time. In total, 18 children worldwide will be recruited to the trial.

Opal is the first patient globally to receive the therapy and is "the youngest globally that's been done to date as far as we know", Bance said.

The gene therapy - DB-OTO - is specifically for children with OTOF mutations. A harmless virus is used to carry the working gene into the patient.

The trial is "just the beginning of gene therapies", Bance said. "It marks a new era in the treatment for deafness."

Martin McLean, a senior policy adviser at the National Deaf Children's Society, said deafness should never be a barrier to happiness or fulfilment. "Many families will welcome these developments, and we look forward to learning about the long-term outcomes for the children treated."

With Opal's hearing restored, her parents now have a fresh problem to contend with: their daughter's new favourite hobby is slamming cutlery on the table to make as much noise as possible.

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UK toddler has hearing restored in world first gene therapy trial

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Opal Sandy can hear almost perfectly after groundbreaking surgery that took just 16 minutes

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Calls for overhaul of gene-technology regulations and wide public discussion

An expert panel set up by Royal Society Te Apārangi to consider the implications of new technologies that allow much more controlled and precise 'editing' of genes, has concluded it's time for an overhaul of the regulations and that there's an urgent need for wide discussion and debate about gene editing within and across all New Zealand communities.

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An expert panel set up by Royal Society Te Apārangi to consider the implications of new technologies that allow much more controlled and precise 'editing' of genes, has concluded it's time for an overhaul of the regulations and that there's an urgent need for wide discussion and debate about gene editing within and across all New Zealand communities.

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Abstract

Gene editing is arguably the most significant recent addition to the modern biotechnology toolbox, bringing both profoundly challenging and enabling opportunities. From a technical point of view the specificity and relative simplicity of these new tools has broadened the potential applications. However, from an ethical point of view it has re-ignited the debates generated by earlier forms of genetic modification. In New Zealand gene editing is currently considered genetic modification and is subject to approval processes under the Environmental Protection Authority (EPA). This process requires decision makers to take into account Māori perspectives. This article outlines previously articulated Māori perspectives on genetic modification and considers the continuing influence of those cultural and ethical arguments within the new context of gene editing. It also explores the range of ways cultural values might be used to analyse the risks and benefits of gene editing in the Aotearoa New Zealand context. Methods used to obtain these perspectives consisted of (a) review of relevant literature regarding lessons learned from the responses of Maori to genetic modification, (b) interviews of selected 'key Maori informants' and (c) surveys of self-selected individuals from groups with interests in either genetics or environmental management. The outcomes of this pilot study identified that while Māori informants were not categorically opposed to new and emerging gene editing technologies a priori, they suggest a dynamic approach to regulation is required where specific uses or types of uses are approved on a case by case basis. This study demonstrates how the cultural cues that Māori referenced in the genetic modification debate continue to be relevant in the context of gene editing but that further work is required to characterize the strength of various positions across the broader community.

Keywords: indigenous, gene editing, Māori, ethics, regulation, New Zealand

Background

Gene Editing

All living organisms contain long molecules of DNA which are inherited between generations. The total sum of DNA from an organism is referred to as its "genome," which itself includes all of its "genes." An organism's DNA affects how it looks and how it behaves. DNA can change spontaneously, generating new "mutations" (or "variants") that can have a visible effect. For example several mutations of the eye color character have occurred during human history. Similarly, key mutations in traits such as grain yield and milk production selected by farmers have contributed to crop domestication and agriculture. In the last century and since the discovery of the structure and importance of DNA as the molecule encoding life, several techniques have been developed to artificially alter genes and genomes. The latest of such technique is "gene editing."

Gene editing is a technology that enables scientists to alter the DNA of an organism in a very precise way. The technique relies on the CRISPR-Cas9 (clustered, regularly interspaced, short palindromic repeats-associated protein Cas9) system that is capable of recognizing a specific DNA motif in the genome. The Cas9 protein then cuts it the DNA sequence to produce double stranded breaks, which can be fixed by the repair system in a non-homologous end joining manner with variable sizes of insertions or deletions and therefore generates DNA mutations (Jinek et al., 2012; Chang et al., 2016). Gene editing has some key differences with other techniques used to generate DNA mutations. Radiation-based mutation using gamma-ray irradiation generates many DNA mutations across the genome in a random fashion. Radiation is used extensively in plant breeding to generate new traits such as seedless table grapes. Unlike radiation, gene editing only targets a precise location in the genome. Another method that has been used in the last 40 years is genetic modification using transgenics (often referred to as GM or GMOs). The principle of transgenics is to insert an entire gene into the host genome. The inserted gene often comes from a different organism. For example, insect-resistance Bt maize, eggplant, and cotton result from inserting a gene from the bacteria Bacillus thuringiensis. Gene editing is being promoted as a more precise technology that could be used to amend an existing gene rather than inserting foreign genes into an organism.

Potential Applications

Gene editing is bringing both profoundly challenging and enabling opportunities for applications in human health, natural resource stewardship and primary production. In medicine, gene editing has already been approved for use in patients to make immune cells attack cancer cells or to mutate HIV virus DNA to stop it from replicating (Tebas et al., 2014; Reardon, 2015). In agriculture, gene editing is being used to create more hardy, nutritious and productive plants and animals (Shan et al., 2013; Wang et al., 2015). In conservation, researchers may be able to use gene editing to introduce a sterility gene into a pest as part of a pest-eradication programme, or spread a malaria resistance gene in mosquitoes (Hammond et al., 2016).

The broad range of potential applications of gene editing has the potential to re-ignite ethical debates generated by earlier forms of genetic modification. In Aotearoa New Zealand, genetically modified organisms (GMO's) have been regulated since the establishment of the Environmental Risk Management Authority (ERMA) through the Hazardous Substances and New Organisms Act ( 1996), responsibilities which transitioned to the Environmental Protection Authority (EPA) through the Environmental Protection Authority Act ( 2011). Early applications of gene technologies to create transgenic organisms were met with public outrage, leading to the establishment of a Royal Commission of Inquiry into Genetic Modification. Over 10,000 public submissions were considered by the Commission in the development of its report, which has led to no genetically modified crops being grown in New Zealand (Royal Commission on Genetic Modification, 2001). Māori were significant contributors to the debates on genetic modification and regulatory processes provide specific recognition of Māori values within decision-making processes for new organisms including GMOs (Cram et al., 2000; Environmental Protection Authority, 2016).

The variability surrounding the regulation of gene editing in the international context has led to the recent establishment of a Royal Society of New Zealand (RSNZ) Gene Editing Panel to engage the public in discussions and provide advice to the New Zealand Government on potential options for regulation (Royal Society of New Zealand, 2016, Royal Society of New Zealand, 2017a, b). Gene editing is currently considered genetic modification and therefore non-human gene-edited organisms are classified as "new organisms" and therefore are subject to approval processes under the EPA, a process which includes the incorporation of Māori perspectives.

Literature Review

The literature suggests there are more Māori positioned on the anti-GM end of the spectrum (Gardiner, 1997; Cram et al., 2000), however a distinction is apparent between GMOs for commercial production with no clear cultural or environmental benefits and those that might provide direct community benefit (Roberts and Fairweather, 2004; Smith et al., 2013). GM projects that had a clear benefit or genuine contribution to communities and the environment were received more positively. The literature provided some consistent messages about the key Māori cultural concepts and values relevant to biotechnology and genetic research. There is a general consensus that whakapapa (genealogy) sits as the key concept for Māori communities. The second most commonly acknowledged cultural value is mauri (life essence), followed by mana (power/authority) and kaitiakitanga (guardianship). A number of other Māori terms are also used in the course of writing about Māori and biotechnology issues such as mātauranga (indigenous knowledge), tikanga (protocols), Papatūānuku (earth mother), and tangata whenua (indigenous people, literally people of the land). Culturally based concepts have also been associated with specific functions. For example, concepts related to "Consultation and Relationships" include kawa (customary principles), tika (right, correct), and manaakitanga (to care for, look after), while tapu (sacred, restricted), taonga (precious), wairua (spirit) are associated with the status of DNA and tākoha (gift) to the sharing of DNA (Beaton et al., 2016; Hudson et al., 2016a). Table 1 highlights the most commonly discussed Māori concepts and values.

Table 1

Concept:

Whakapapa

Mauri

Kaitiakitanga

Mana

This article outlines previously articulated Māori perspectives on genetic modification, then considers the continuing influence of those cultural and ethical arguments within the context of recent developments in gene editing, and finally explore with key Māori informants how cultural values might be used to analyse the risks and benefits of gene editing in the Aotearoa New Zealand context.

Methods

This project is a part of a New Zealand government funded research programme led by Plant & Food Research Ltd on "Turbo-breeding for New Zealand's plant industries" primarily focusing on the adoption of new breeding technologies in the horticulture sector. A component of the project explores the co-innovation interface, the interface of cultural and commercial interests and concerns, with a view to identifying processes that support Māori organizations to participate in research and commercialization activities involving gene editing technologies. The aim of this part of the project was to assess the on-going relevance of Māori concepts in the context of gene-editing. Data was collected through three key activities; a literature review; key informant interviews; and an electronic survey.

A review of 38 key publications between 2005 and 2017 provided the foundation for this project. The review covered literature relating to Māori and biotechnology, genetic modification, and genetic research with a particular focus on the Māori values, concepts and perspectives that have previously been articulated. The review became the basis for a discussion document, Māori Perspectives & Gene Editing: A Discussion Paper (Mead et al., 2017), which informed preliminary discussions with a number of agencies and Māori networks, such as the EPA's Ngā Kaihautu Tikanga Taiao (Māori Advisory Body), Te Herenga Network (National Māori Network of Iwi Environmental Practitioners), Te Tira Whakamataki (the Māori Biosecurity Network), the Biological Heritage National Science Challenge, and public consultation exercises on gene editing led by the Royal Society of New Zealand.

Ethics Approval was gained from the University of Waikato's delegated research ethics committee within the Faculty of Māori and Indigenous Studies for the collection of data from key informant interviews and an electronic survey which used the same set of open questions. The questions emerged from the key concepts identified in the literature review and focused on the potential applications and opportunities associated with gene editing, the key issues and concerns associated with gene editing, whether gene editing should be considered the same as genetic modification, and the relevance of key Māori values and concepts ( Table 1 ) to understanding gene editing.

Eight key informants (2 × males, 6 × females) were purposefully selected from the researchers networks with interests in plant health, environmental health, human health, business, research, public understanding, and public policy to provide a range of informed Māori perspectives. Four of the key informants are active researchers albeit not in genomic sciences, and the others have strong relationships with researchers and communities. They were chosen because of the roles they play as translators between science teams and Māori communities and general familiarity with both scientific research and Māori perspectives. Key informants were interviewed separately and an electronic survey was shared with members of the Te Herenga Network (National Māori Network associated with the Environmental Protection Authority) and the SING Alumni Network (Summer Internship for Indigenous Genomics programme). The survey, which resulted in nine additional responses, was used to broaden the range of perspectives and reduce the potential bias associated with the key informant interviews. All participants were Māori and the responses to each question in the interviews (KI) and the survey (SR) were grouped and analyzed manually by the researchers using guided thematic analysis (Coffey and Atkinson, 1996). The process of coding empirical material to the research questions and emerging themes, was conducted across key domains, including potential benefits, concerns, and the relevance of Māori values and concepts.

Results

Interview and Survey Responses

What Do You See Are the Potential Applications/Opportunities Associated WITH GENE Editing?

Participants saw different opportunities for gene editing to support their communities aspirations in horticulture, conservation, maintaining the health and biodiversity of the environment, or to address human health issues. A range of potential applications were identified including preservation of endangered species of plants and animals, new health related therapies, protecting biodiversity, creating health and food security, sequencing of rare threatened and endangered endemic species (and their medical chemotypes), human health, environmental restoration, sustainable enterprise, pest control, and pest eradication. One participant noted a primary interest as ensuring that gene editing was stopped.

What Do You See Are the Key Issues/Concerns That Arise From Use of Gene Editing?

Participants concerns about the use of gene editing centered on the risks of adopting this technology. The risk of unintended consequences, whether it is possible to do rigorous assessments of the potential downstream effects, the reversibility of any genetic modifications, ethical considerations, and effect on kaitiaki (guardians) responsibilities were highlighted. Participants identified an innate risk from a cultural perspective that needs to be managed to limit unethical or unauthorized modifications. A number of the participants expressed a view that mixing genetic material from different species is unnatural and there was also a degree of anxiety about editing an organisms genome especially for economic gain.

An issue was raised about the benefits to society and concerns that technology favors the wealthy and tends to increase inequities through the commodification of resources. Some participants had concerns about the use of gene editing in the environment and others were more concerned about its use in humans. Specific issues were raised in relation to editing genes in the human germline because it is passed down to future generations and there is no information about the long-term effects. Risks to the environment were associated with the release of modified organisms into the environment where commercial interests exclude wider community benefits. Concerns were also expressed about the level of experimentation required before benefit emerges and how the community are kept up to date with what types of activities are underway. Fostering public conversations about genetic modification are necessary as public understanding lags well behind the current state of expert knowledge and technical capability.

In NZ law, gene editing is considered the same as genetic modification. In other countries gene editing is treated differently from GM. Do you think we have the right legal approach in NZ or do you think the law should be amended in light of the developing technology?

Strictly speaking gene-editing is a form of genetic modification and while some participants considered gene editing and genetic modification to be the same thing, others saw a spectrum of gene technologies.

"This highlights an issue regarding terminology and general understanding of genetic technologies. Genetic modification encompasses a spectrum of technologies with transgenics on one end and modern gene-editing on the other." (Survey Response, SR7)

It was generally accepted that a regulatory regime should cover both Genetic Modification and Gene editing as a precautionary approach was necessary. The level of regulation ranged from " No GMO in New Zealand"(SR5) to " Only in the laboratory and total containment" (SR6) to " after strict substantive and procedural decision-making" (SR4). Participants recognized the inconsistencies arising from the treatment of all genetically modified organisms in the same way and while there was some sympathy for the differences between technologies, the status quo allows all gene technologies to be monitored appropriately. Some of the participants felt that inter- and intra-specific genetic modification should be treated differently and possibly on a case by case basis. However, any change would require more consultation with Māori and the wider public to assess the effect on Māori rights and interests.

Do You Think Gene Editing Can Support Kaitiaki Responsibilities and Under What Circumstances?

Gene editing technologies are potentially one tool in the toolbox to protect and save species or be used to enhance health. Considerations will include the intent of the use, how kaitiaki understand the science, and whether its use disrupts or enhances the relationships they have with taonga species.

"Values-based organisations can use technologies to support their aspirations and in that respect they have a duty to explore all avenues that support kaitiaki responsibilities for taonga species." (Key Informant; KI1)

The participants generally felt that gene editing could support kaitiaki to exercise their responsibilities and in some situations will be forced to consider extreme options like gene-editing to deal with intractable "wicked problems," where all the choices appear on a spectrum of ethically challenging options. This might arise in terms of pest control as a tool to protect and enhance wildlife or as a way of correcting a variant of a gene known to be responsible for a disease. Where species extinctions are occurring, kaitiaki might explore gene-editing as an option. These decisions would be by hapū (sub-tribes) or iwi (tribes) as to whether this is an appropriate technology to support their responsibilities.

Do You Think Whakapapa Is Affected if You Introduce DNA Into One Species From Another Species? Is This the Same Case if You Edit DNA Within the Same Species?

All the participants thought that whakapapa was affected by introducing DNA from one species into another through genetic modification. Some kaumatua (elders) are against interspecies transfer while others are less concerned, and the participants expressed mixed opinions in relation to the effect of gene editing on whakapapa. Some participants felt it was dependent on the extent of the edit as some variation within the same species or sub-species is expected. The effect on whakapapa was also thought to be connected to the relationality between the species sharing or exchanging DNA. Where DNA associated with genes that are shared in different species is exchanged, this will have less impact on whakapapa. However, if a transferred gene does not have a naturally occurring sequence, then this could be seen to be cutting across whakapapa links. Some felt that gene editing definitely affects whakapapa but that could also be in a positive direction.

"Altering genes changes the genetic make-up of an individual but can be viewed similarly to an organ transplant. Whether it is ethical to change or introduce DNA into a species is another thing. For me whakapapa is lineage and your ties to whanau and your ancestors and that doesn't change with the introduction of foreign DNA. It could be viewed as enhancing your lineage to some or diluting it to others." (SR8)

Is There a Difference Between Applying Gene-Editing for "Taonga Species" and Introduced or Commercially Produced Species?

There were mixed views on whether there was a difference when applying gene editing to a taonga (precious) species or an introduced or commercially produced species. It was clear that Māori should have a say in relation to both but the difference arose from the nature of the relationship Māori have with taonga species through Treaty of Waitangi obligations and indigenous rights.

"Māori should have the final say for approving editing in taonga species. For introduced or commercially produced species, all groups in NZ should be consulted (including Māori)." (SR3)

"The only difference between gene editing in taonga species and introduced or commercial species is that our responsibility to taonga species means that there is a much greater impetus to ensure minimal disruption to the whakapapa, mana, and mauri of these species."(SR7)

Some felt that all species are interdependent and therefore taonga but would need to consider their different degrees of importance and how to deal with hybridity. Exotic species have been incorporated within rongoa Māori (traditional medicine) formulations since colonization and as such Māori walk in two worlds. Regardless of whether gene editing was being used for taonga species or other species the risks involved and ethical considerations are the same.

Is Mauri of a Species/Person Affected if the Gene-Edit Mimics a Natural Mutation/Variant?

The effect on mauri (life essence) represents one of the key moral dilemmas associated with genetic modification. Most people believe the life force is changed by gene editing but there were variations on this theme. Some felt that mauri was not affected if the change occurs naturally, and others mentioned that the effect on mauri can be both positive and negative. The effect on the mauri is related to the nature and size of the change including the heritability of the new characteristics. There was a concern expressed around the unintended effects of genetic variation and whether that changes the long term resilience of a species.

"Naturally occurring mutations/variants often occur due to environmental changes which enable adaptation to occur in the species. I feel a gene edit can either enhance or reduce Mauri depending on the phenotypic outcome of that species." (SR8)

If mana is recognised through Māori leading the project and the research objectives are to benefit Māori whanau (families), hapū (sub-tribe), or iwi (tribe) are the same concerns still relevant?

There was a general belief amongst the participants that enhancing mana through Māori leadership would increase the level of engagement and acceptance to a project involving gene editing however the same concerns about gene editing still apply. There was a feeling that all gene editing, whether it be for conservation, commercial, or indigenous interests, should be subject to standardized processes even though cultural protocols are specific to each tribal authority.

"I don't believe it is necessary for Māori to lead a project, but it is critical that Māori are at least partners, to ensure that Mana is recognised." (SR7)

Participants thought it was important to define the space and reasoning for using gene editing technology including how the project and any data/intellectual property would be managed. Māori input into this process is necessary especially for taonga species and Māori should also consider the impact of our decisions on other indigenous peoples, especially if the intended use is to eradicate a species endemic to another country.

Discussion

A small but significant portion of research exists concerning indigenous peoples' responses to bio/nano-technology, generally establishing the 'indigenous position' as one strongly against these developments and their commercialization (Gardiner, 1997; Harry et al., 2000; Leier, 2002; Reynolds and Smith, 2002; Hutchings and Reynolds, 2005; Mead and Ratuva, 2007). The prevailing critique has been that most 'bio/nanotechnology projects are inconsistent with Māori values, impinge on Māori rights and sovereignty, and continue a process where indigenous cultures, values, knowledge systems and even lives are marginalized and undervalued (Cram et al., 2000; Roberts and Fairweather, 2004; Cram, 2005; Hutchings and Reynolds, 2005; Te Momo, 2005; Hutchings, 2009).

Despite inclusion in existing regulatory processes and more positive interactions over the past decade (Hemara, 2006; Cheung et al., 2007; Te Momo, 2007; Hudson et al., 2012, 2016c) and the responses of participants in this project, a widespread social license for the use of gene-based technologies amongst the Māori community is unlikely in the short term. Generally, Māori do not oppose new and emerging gene editing technologies a priori, but instead raise concerns as to how the technologies should be used and the rationale, objectives and consequences of choosing them. Individual subjectivities inform the process as personal preferences for particular technologies are grounded in their own values, experiences and knowledge (Te Momo, 2007; Smith et al., 2016). The experience of the participants played an interesting part in the identification and management of potential risk. Sometimes those with backgrounds in particular fields, for example the environment, were more comfortable with its potential application in that domain and highlighted risks associated with other areas like health. In other cases, the reverse was true where experience in a field highlighted the specific concerns for application in that domain. The general discomfort all the participants expressed was reflected in the desire for appropriate regulation and a sense that there will always be justifiable use-cases and unpalatable use-cases. This anticipates a more dynamic approach to regulation where specific uses or types of uses are approved on a case by case basis.

Māori participation in discussions on gene technologies is as much cultural and political as scientific [(Cram, 2005), p. 62; (Hudson et al., 2010; Smith et al., 2013)]. Discussions on gene-based technologies cannot be divorced from discourse on land ownership and control over natural resources and debates traverse the spectrum of philosophical, social, ethical, and technical dimensions (Smith et al., 2013). Māori perspectives on biotechnology/genetic technologies frequently reference core cultural concepts as conceptual markers, derived from mātauranga Māori (indigenous knowledge) and tikanga Māori (Māori values), which are intrinsic to an indigenous way of viewing and living in the world. These cultural cues provide the basis for describing the cultural logic that underpins engagement in a culturally acceptable manner (Cram et al., 2000; Hudson et al., 2010, 2016c; Smith et al., 2013). This research demonstrates the cultural cues that Māori referenced in the genetic modification debate, and subsequent conversations about biotechnologies, continue to be relevant in the context of gene editing. The Māori concepts of whakapapa (genealogy), mauri (life essence), mana (authority), and kaitiakitanga (guardianship) feature prominently. Whakapapa and mauri relate to the organism itself and mana and kaitiakitanga refer to the relationship that people have with that organism. Whakapapa is a key reference point when talking about genetics or genomics (Hudson et al., 2016a) because it provides the foundation for how Māori construct their identities and their relationships with other species (Roberts, 2005, 2013; Hudson et al., 2007). Mauri relates to the distinctive and special nature of an organism including its right to life (Hudson et al., 2010; Mead, 2017). Mana relates to authority and provides a responsibility to act in the interests of the broader community (Mead, 2017). The expression of kaitiakitanga enhanced through the recognition of mana whenua status presupposes that Māori have authority over their lands and resources and that the use of gene technologies is done in ways that supports these outcomes (Thompson, 2018).

Participants in this study suggested that the effect of gene editing on Māori values is not always in a negative direction and it was suggested that whakapapa, mana, mauri, and kaitiakitanga might be enhanced through the use of gene editing technologies. This suggests that values based frameworks developed for other gene based technologies (Wilcox et al., 2008; Hudson et al., 2016c) will remain relevant in for gene editing applications. What the enhancement or diminishment of these Māori values might look like is summarized in Table 2 .

Table 2

Value/Concept

Value enhancement

Value diminishment

According to New Zealand law, gene editing is not deemed distinct, rather it is seen as one of many processes, tools, methods, or products of genetic modification and as such is subject to the same regulations as any other GMO. A key issue here is whether it makes sense to regulate a technology rather than regulating the outcome or product of the technology. Gene editing will allow the generation of outcomes/products similar, or identical to those generated by technologies not covered by legislation. Gene editing does not necessarily insert foreign DNA into the genome of the host organism and the DNA mutations resulting from gene editing involve small DNA sequence changes that cannot be differentiated from natural ones, even using modern sequencing technologies. The RSNZ is currently engaging the New Zealand public in debates about gene editing through a series of discussion documents (Royal Society of New Zealand, 2016; Royal Society of New Zealand, 2017a, b) and a public speaker series. The topic has recently been brought to back into the spotlight by comments from the outgoing NZ Chief Government Science Advisor who suggested that while the use of gene technologies continued to be heavily debated, from a scientific point of view " There are no significant ecological or health concerns associated with the use of advanced genetic technologies," and that we need to engage society in debate that is " more constructive and less polarized than in the past." (Science Media Centre, 2018) ( https://www.sciencemediacentre.co.nz/2018/07/02/changing-of-the-chief-scientist-guard-in-the-news/). The participants in this study wanted to engage in a constructive discussion to create a robust regulatory framework that addresses gene editing on a case by case basis and utilizes Māori values within the decision-making process.

Summary

Gene editing is the most recent gene based technology promising benefits across health, environmental, and commercial domains. It emerges in the wake of decades old, ethically charged, debates about GMOs and transgenic applications which seared controversy about gene technologies into the public consciousness. As the New Zealand government considers whether to change the regulations around gene editing technologies it is supporting a new round of public consultation exercises. While Māori have expressed strong views and opposition to genetically modified organisms in the past, it is important to assess the continuing influence of those perspectives.

The participants demonstrated that Māori values and cultural concepts continue to inform Māori perspectives on biotechnology and their regulation. Whakapapa, mauri, mana, and kaitiakitanga provide a cultural scaffold for considering the philosophical, moral, ethical and technical dimensions relevant to the use of gene editing technologies. It is apparent that a range of views exist across the Māori community and that participants are prepared to consider the use of gene editing on a case dependent basis, especially where it aligns with Māori worldviews. Incorporating Māori values into decision-making processes could provide a balancing factor to ensure broader community interests remain a key consideration in the future use of gene editing technologies. The application of gene editing technologies heightens societal sensitivities about inequities as their use tends to prioritize commercial interests over community benefit (Smith, 2016). However, further research is required to characterize the strength of the various positions identified in this pilot study and to explore its relevance to other indigenous communities.

Author Contributions

MH: contribution to framing and writing, analysis of surveys and interviews, primary editor. AM: primary data collector, contribution to framing and writing, analysis of literature, and development of tables. DC: contribution to framing and writing of scientific context, review of analysis and manuscript. NR, SM, and PW: data collection, review of analysis and manuscript. AA: contribution to writing of scientific context, review of analysis and manuscript, principal investigator.

Conflict of Interest Statement

DC and AA are employed by The New Zealand Institute for Plant and Food Research. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors wish to acknowledge the Ministry of Business, Innovation and Employment for research funding (C11X1602) and survey/interview participants for sharing their knowledge.

Glossary

Ngā Kaihautu Tikanga Taiao Māori advisory body for Environmental Protection Authority

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Articles from Frontiers in Bioengineering and Biotechnology are provided here courtesy of Frontiers Media SA

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Indigenous Perspectives and Gene Editing in Aotearoa New Zealand

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Gene editing is arguably the most significant recent addition to the modern biotechnology toolbox, bringing both profoundly challenging and enabling opportunities. From a technical point of view the specificity and relative simplicity of these new tools ...

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Level 3 Biology: Methods of genetic manipulation

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View LearnCoach’s step-by-step video playlist. The playlist includes teaching videos, practice questions, and model answers for Level 3 Biology: Methods of genetic manipulation

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Centers for Disease Control and Prevention

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Your genes play an important role in your health, but so do your behaviors and environment, such as what you eat and how physically active you are. Epigenetics is the study of how your behaviors and environment can cause changes that affect the way your genes work. Unlike genetic changes, epigenetic changes are reversible and do not change your DNA sequence, but they can change how your body reads a DNA sequence.

Gene expression refers to how often or when proteins are created from the instructions within your genes. While genetic changes can alter which protein is made, epigenetic changes affect gene expression to turn genes "on" and "off." Since your environment and behaviors, such as diet and exercise, can result in epigenetic changes, it is easy to see the connection between your genes and your behaviors and environment.

How Does Epigenetics Work?

Epigenetic changes affect gene expression in different ways. Types of epigenetic changes include:

DNA Methylation

DNA methylation works by adding a chemical group to DNA. Typically, this group is added to specific places on the DNA, where it blocks the proteins that attach to DNA to "read" the gene. This chemical group can be removed through a process called demethylation. Typically, methylation turns genes "off" and demethylation turns genes "on."

Histone modification

DNA wraps around proteins called histones. DNA wrapped tightly around histones cannot be accessed by proteins that "read" the gene. Some genes are wrapped around histones and are turned "off" while some genes are not wrapped around histones and are turned "on." Chemical groups can be added or removed from histones and change whether a gene is unwrapped or wrapped ("on" or "off").

Non-coding RNA

Your DNA is used as instructions for making coding and non-coding RNA. Coding RNA is used to make proteins. Non-coding RNA helps control gene expression by attaching to coding RNA, along with certain proteins, to break down the coding RNA so that it cannot be used to make proteins. Non-coding RNA may also recruit proteins to modify histones to turn genes "on" or "off."

How Can Your Epigenetics Change?

Your epigenetics change as you age, both as part of normal development and aging and in response to your behaviors and environment.

Example: Nerve cell vs. Muscle cell

Your muscle cells and nerve cells have the same DNA but work differently. A nerve cell transports information to other cells in your body. A muscle cell has a structure that aids in your body's ability to move. Epigenetics allows the muscle cell to turn "on" genes to make proteins important for its job and turn "off" genes important for a nerve cell's job.

Example: Study of newborn vs. 26-year-old vs. 103-year-old

DNA methylation at millions of sites were measured in a newborn, 26-year-old, and 103-year-old. The level of DNA methylation decreases with age. A newborn had the highest DNA methylation, the 103-year-old had the lowest DNA methylation, and the 26-year-old had a DNA methylation level between the newborn and 103-year-old ( 1).

Example: Smokers vs. non-smokers vs. former smokers

Smoking can result in epigenetic changes. For example, at certain parts of the AHRR gene, smokers tend to have less DNA methylation than non-smokers. The difference is greater for heavy smokers and long-term smokers. After quitting smoking, former smokers can begin to have increased DNA methylation at this gene. Eventually, they can reach levels similar to those of non-smokers. In some cases, this can happen in under a year, but the length of time depends on how long and how much someone smoked before quitting ( 2).

Epigenetics and Health

Epigenetic changes can affect your health in different ways:

Example: Mycobacterium tuberculosis

Mycobacterium tuberculosis causes tuberculosis. Infections with these germs can cause changes to histones in some of your immune cells that result in turning "off" the IL-12B gene. Turning "off" the IL-12B gene weakens your immune system and improves the survival of Mycobacterium tuberculosis ( 3).

Cancer
Certain mutations make you more likely to develop cancer. Likewise, some epigenetic changes increase your cancer risk. For example, having a mutation in the BRCA1 gene that prevents it from working properly makes you more likely to get breast and other cancers. Similarly, increased DNA methylation that results in decreased BRCA1 gene expression raises your risk for breast and other cancers (4).While cancer cells have increased DNA methylation at certain genes, overall DNA methylation levels are lower in cancer cells compared with normal cells. Different types of cancer that look alike can have different DNA methylation patterns. Epigenetics can be used to help determine which type of cancer a person has or can help to find hard to detect cancers earlier. Epigenetics alone cannot diagnose cancer, and cancers would need to be confirmed with further screening tests.

Example: Colorectal Cancer

Colorectal cancers have increased methylation at the SEPT9 gene. Some commercial epigenetic-based tests for colorectal cancer look at DNA methylation levels at the SEPT9 gene. When used with other diagnostic screening tests, these epigenetic based tests can help find cancer early ( 5)( 6).

Nutrition During PregnancyA pregnant woman's environment and behavior during pregnancy, such as whether she eats healthy food, can change the baby's epigenetics. Some of these changes can remain for decades and might make the child more likely to get certain diseases.

Example: Dutch Hunger Winter Famine (1944-1945)

People whose mothers were pregnant with them during the famine were more likely to develop certain diseases such as heart disease, schizophrenia, and type 2 diabetes ( 7). Around 60 years after the famine, researchers looked at methylation levels in people whose mothers were pregnant with them during the famine. These people had increased methylation at some genes and decreased methylation at other genes compared with their siblings who were not exposed to famine before their birth ( 8)( 9)( 10). These differences in methylation could help explain why these people had an increased likelihood for certain diseases later in life ( 7)( 10)( 11)( 12).

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office of genomics and precision public health

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Your genes play an important role in your health, but so do your behaviors and environment, such as what you eat and how physically active you are. Epigenetics is the study of how your behaviors and environment can cause changes that affect the way your genes work.

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Cardiac Comprehensive Kit analyzes 292 genes and covers major inherited cardiovascular disorders.
Genes Tested
AARS2, ABCA1, ABCC6, ABCC9, ABCG5, ABCG8, ACAD9, ACADVL, ACTA1, ACTA2, ACTC1, ACTN2, ACVR1, ACVR2B, ACVRL1, ADAMTS2, AFF4, AGK, AKAP9, AKT3, ALDH18A1, ALMS1, ALPK3, ANK2, ANKRD1, APOA5, APOB, APOE, ATP6V0A2, ATP6V1A, ATP6V1E1, B3GALT6, B4GALT7, BAG3, BGN, BMPR1B, BMPR2, BRAF, C1R, C1S, CACNA1C, CACNA2D1, CACNB2, CAD, CALM1, CALM2, CALM3, CASQ2, CAV1, CAV3, CBL, CBS, CFAP53, CCND2, CDK13, CELSR1, CELSR2, CELSR3, CHD4, CHD7, CHST14, CITED2, COA5, COA6, COL1A1, COL1A2, COL3A1, COL4A1, COL5A1, COL5A2, COX15, CPT2, CREBBP, CRELD1, CRYAB, CSRP3, CTNNA3, DES, DMD, DNAH11, DNAH5, DNAH6, DNAI1, DNAJC19, DOLK, DSC2, DSE, DSG2, DSP, DTNA, EFEMP2, EHMT1, EIF2AK4, ELAC2, ELN, EMD, ENG, EPHB4, EVC, EVC2, FBLN5, FBN1, FBN2, FHL1, FKBP14, FKTN, FLNA, FLNC, FOXC1, FOXE3, FOXH1, GAA, GANAB, GATA4, GATA5, GATA6, GATAD1, GBE1, GDF1, GJA5, GLA, GNAI2, GORAB, GPC3, GPD1L, GTPBP3, HADHA, HADHB, HAND1, HAND2, HCN4, HRAS, ILK, JAG1, JPH2, JUP, KARS1, KCNA5, KCNAB2, KCND3, KCNE1, KCNE2, KCNE3, KCNH2, KCNJ2, KCNJ5, KCNJ8, KCNK3, KCNQ1, KDM5B, KMT2D, KRAS, LAMA4, LAMP2, LDB3, LDLR, LDLRAP1, LIPA, LMNA, LOX, LPL, LTBP4, LZTR1, MAP2K1, MAP2K2, MED13L, MFAP5, MIB1, MMP21, MRAS, MRPL3, MRPL44, MTO1, MYBPC3, MYH11, MYH6, MYH7, MYL2, MYL3, MYL4, MYLK, MYLK2, MYOZ2, MYPN, NDUFB11, NDUFV2, NEBL, NEXN, NF1, NF2, NIPBL, NKX2-5, NKX2-6, NME7, NODAL, NOTCH1, NOTCH2, NPPA, NR2F2, NRAS, PCSK9, PDLIM3, PIK3CA, PIK3R2, PITX2, PKD1L1, PKP2, PLD1, PLN, PLOD1, POGZ, PPA2, PPP1CB, PRDM16, PRDM5, PRDM6, PRKAG2, PRKD1, PTPN11, PYCR1, RABGAP1L, RAF1, RASA1, RASA2, RBFOX2, RBM10, RBM20, RIT1, RRAS, RYR2, SALL4, SASH1, SCN10A, SCN1B, SCN2B, SCN3B, SCN4B, SCN5A, SCO2, SEMA3D, SEMA3E, SGCD, SHOC2, SKI, SLC22A5, SLC25A20, SLC25A3, SLC25A4, SLC2A10, SLC39A13, SMAD3, SMAD4, SMAD6, SMAD9, SMARCB1, SNTA1, SOS1, SOS2, SPRED1, STAMBP, TAB2, TAFAZZIN, TBX1, TBX20, TBX4, TBX5, TCAP, TECRL, TFAP2B, TGFB2, TGFB3, TGFBR1, TGFBR2, TK2, TLL1, TMEM260, TMEM43, TMEM70, TNNC1, TNNI3, TNNT2, TPM1, TRDN, TRPM4, TSFM, TTN, TTR, VCL, ZEB2, ZFPM2, ZIC3, ZNF469

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Why Genetic Testing | Medicover Genetics

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Cardiac Comprehensive Kit analyzes 292 genes and covers major inherited cardiovascular disorders.

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One of the reasons human biology is so interesting is because it's so complex; rarely are there neat answers.

We're often taught that when it comes to biological sex, everything fits into two boxes: XY and XX. However, it turns out that, just like the rest of biology, things are a little more complicated than that.

Some people not have chromosomes or sexual characteristics that neatly fit into those two boxes. These people have disorders of sex development (DSDs), also known as differences of sex development or intersex conditions. Due to the taboo nature of the subject and differing opinions of what qualifies as a DSD, it's hard to pin down exactly how many people have DSDs-estimations have ranged between 0.05% and 1.7% of the world's population.

Before we start discussing the details, let's define some terms that often come up in discussions of sex:

Sex determination refers to the chromosomes an individual has-typically XX or XY.

Sex differentiation refers to the anatomical development of reproductive systems-for example, people with XX chromosomes tend to have cervixes, vaginas, uteruses, etc.

Gender refers to the " norms, behaviors, and roles " typically associated with femininity and masculinity. Gender pertains more to culture than biology, so we won't discuss it here.

Those definitions limit humans to one of two options, but as we said before, biology tends to be complicated !

Today on the Visible Body Blog, we're going to discuss sex chromosomes, genes, and DSDs.

Processes of sex determination and differentiation

Let's start at the beginning: spermatogenesis and oogenesis. During the process of meiosis, specialized sex cells (spermatogonia or oogonia) duplicate themselves and divide to create gametes (sperm or eggs).

GIF of meiosis I model from Visible Biology.

Sperm and eggs each have one set of chromosomes-but sometimes those sets aren't the typical 23 chromosomes. This is how people can inherit a combination of X and Y chromosomes other than the usual XX or XY. We'll touch on this more later!

During fertilization, the sperm and egg fuse to form a zygote, which divides into more cells to become an embryo.

The egg carries an X chromosome , and the sperm can carry X or Y. The Y chromosome carries 55 genes compared to the X's 900+, and one of the Y chromosome's genes is the SRY gene. The SRY gene provides instructions for making the sex-determining region Y protein. This protein typically causes the fetus to develop testes.

Remember how we said that the SRY gene is carried on the Y chromosome? Well, it's possible for X and Y chromosomes to swap some DNA, causing the X chromosome to carry the SRY gene.

At six weeks of development, the fetus's genes dictate which gonads develop. More than 25 genes contribute to sex differentiation !

Fetuses with XY chromosomes typically develop testes, and those testes produce hormones that spur the development of male sex organs, while fetuses with XX chromosomes typically develop female sex organs. However, that's not always the case.

Typical reproductive system anatomy from Human Anatomy Atlas 2022 +.

DSDs

Let's take a quick look at a few DSDs, including two of the most common disorders, trisomy X and Klinefelter syndrome.

It's important to note that not all DSDs are a result of a karyotype (an individual's set of chromosomes) outside of XX and XY. Like we mentioned before, more than 25 genes contribute to sex differentiation: mutations of these genes can all alter sex differentiation, which means that there is a wide range of DSDs.

Trisomy X

Trisomy X , also known as XXX syndrome, occurs when a person has three X chromosomes. Trisomy X is caused by nondisjunction , which happens when the X chromosomes fail to separate properly. Nondisjunction occurs during anaphase of mitosis or either stage of meiosis. Trisomy X is usually caused by nondisjunction during meiosis.

Trisomy X occurs in 1 in 1,000 female births. It's estimated that only 10% of people with trisomy X are diagnosed, since symptoms can be mild or not present at all.

Physical characteristics associated with trisomy X include:

Above average height

Epicanthal folds

Clinodactyly (a bent or curved finger)

Wide-spaced eyes

Decreased muscle tone (hypotonia)

People with trisomy X sometimes have medical problems like:

Seizures

Autoimmune conditions

Premature ovarian failure

People with trisomy X have a higher rate of motor and speech delays as children, learning disabilities, anxiety, and depression.

Klinefelter syndrome (KS)

Klinefelter syndrome, also known as 47, XXY, is the most common DSD, occurring in 1 in 500 male births .

Like trisomy X, nondisjunction typically causes KS . It can result from nondisjunction of the maternal or paternal chromosomes; maternal nondisjunction can occur during either stage of meiosis and paternal nondisjunction occurs during meiosis II.

People with KS tend to have the following characteristics:

Taller than average

Sparse body and facial hair

Small phallus and testes

A low-to-average testosterone level

Intention tremors

Overweight

KS is associated with an increased risk of diabetes, autoimmune diseases, and hypothyroidism, as well as an increased risk of learning disabilities and verbal and communication delays and difficulties.

Experts say that up to two thirds of KS cases go undiagnosed, and diagnosis doesn't usually occur until the person experiences fertility problems as an adult. The vast majority of people with KS are considered infertile, but thanks to assisted reproductive techniques , it's possible for them to have children.

Swyer syndrome

People with Swyer syndrome have XY chromosomes and functional female genitalia. However, they lack ovaries.

The cause of Swyer syndrome is unknown, and several genes may be involved .

Because they do not have ovaries to produce sex hormones, people with Swyer syndrome will not go through puberty unless they have horomone replacement therapy. Swyer syndrome usually goes undiagnosed until the teenage years, when puberty and menstruation do not occur. With hormone replacement therapy, puberty will develop normally.

People with Swyer syndrome tend to be taller than average, have a small uterus, and an enlarged clitoris. They can become pregnant through an egg donation. Notably, about 30% of people with Swyer syndrome develop a tumor in the scar tissue that develops where the ovaries would be.

Turner syndrome

Turner syndrome occurs when a person has a single X chromosome (monosomy X) or one whole X chromosome and one partial X chromosome. The exact cause of Turner syndrome is unknown.

Turner syndrome karyotype. Note the single x chromosome. Photo from Wikipedia Commons.

People with Turner syndrome may have

A webbed neck

Heart defects

Early loss of ovarian function

Short stature

Skeletal malformations

A low hairline at the back of the neck

Swollen hands and feet

Some symptoms are connected to the SHOX gene, which helps regulate other genes. Because the protein SHOX encodes contributes to the growth and maturation of the skeleton, missing one SHOX gene on the missing or incomplete X chromosome results in short stature.

Many individuals with Turner syndrome need hormone replacement therapy to start puberty. Congenital heart defects are a concern for people with Turner syndrome, as they increase the risk of complications like high blood pressure and aortic dissection.

Androgen insensitivity syndrome

People who have androgen insensitivity syndrome (AIS) have XY chromosomes and are resistant to androgens, the sex hormones that influence the development of male organs.

People with complete AIS have female external genitalia and undescended testes. They do not have uteruses. They may have ...

Partial closing of the outer vagina

A short vagina

No cervix

An enlarged clitoris

People with partial AIS, where the body only partially resists androgens, can have typically female, typically male, or ambiguous genitalia.

Mosaicism

The last condition we'll talk about is mosaicism. Unlike the other syndromes we've covered, mosaicism isn't a DSD, but it can be connected to a DSD.

Mosaicism is when a person has two or more genetically different sets of cells. It's likely caused by an error in mitosis wherein a cell doesn't split evenly into two cells, creating cells with different numbers of chromosomes. Experts actually think that mosaicism occurs at a small level in most people.

Mosaicism is tied to several DSDs. For example, mosaic Klinefelter syndrome means that some of the cells have XXY chromosomes and some have XY chromosomes. Mosaic KS tends to have fewer symptoms than non-mosaic KS.

Read more

DSDs have long been stigmatized and misunderstood-including by the medical community. If you'd like to learn more, check out these articles.

Be sure to subscribe to the Visible BodyBlog for more anatomy awesomeness!

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Beyond Just XX or XY: The Complexities of Biological Sex

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We're often taught that when it comes to biological sex, everything fits into two boxes: XY and XX. However, it turns out that it's more complicated.

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As a pivotal advance in the gene-editing field and timeline, CRISPR continues to be utilized for research on stem cells and human diseases.

Today, I will explain some of the most notable recent findings in the stem cell-CRISPR field.

To start things off, I have also created an infographic that briefly explains what CRISPR-Cas9 is, what stem cells are, and how they both can be used together to analyze human disease through genetics and potentially treat some specific diseases in the future.

You may also find this review paper on the use of gene-editing in stem cells by Dr. Knoepfler and postdoc Michael Chen to be useful.

A refresher on how CRISPR gene-editing works

CRISPR-Cas9, is a genome-editing mechanism known to be faster, cheaper, and more efficient than previous editing technologies like zinc finger nucleases. It was derived from a natural anti-viral defense system found in bacteria and consists of two main parts-a guide RNA and the Cas9 enzyme-that introduce new sequences into a genome.

The targeted genome could be anything from microbes to plants to humans.

Now on to some specific examples of recent CRISPR gene editing research in stem cells.

Engineered "Invisible" Pluripotent Stem Cells

Scientists at the University of California, San Francisco (UCSF) have genetically engineered pluripotent stem cells that are essentially undetected by the immune system and, therefore, can prevent the obstacle of stem cell transplant rejections. Because cell transplants have the possibility of being seen as foreign and harmful by the immune system, there is a risk of triggering a potent immune response that can harm patients. To avoid this complication and tackle the challenges of induced pluripotent stem cell (iPSC) technology, including cost, genomic instability, and uncertainty of clinical use, UCSF researchers considered designing "universal" iPSCs for any patients.

CRISPR-Cas9 was utilized to alter these IPSCs. The researchers modified three genes in iPSCs, including deleting two genes that control major histocompatibility complexes (MHCs). MHCs are proteins on cell surfaces that display peptides for immune cell recognition, and transplanted organs, tissues, and cells can carry the donor's antigens that differ from the patient's antigens and, thus, MHCs can play key roles in evoking a strong rejection reaction.

The team also found a surface protein called that tells macrophages to not eat (or "phagocytose") the CD47-displaying cells. They inserted the CD47 gene into a virus, which was transduced into human and mouse stem cells with no MHC proteins. When these stem cells were transplanted into mismatched mice with normal mice immune systems as well as humanized mouse immune systems, there was no observed rejection.

As a result of these modifications, differentiated cells made from the gene-edited iPSCs may avoid detection by human recipients' immune systems after transplantation in potential future clinical trials. However, such transplanted cells that are largely invisible to the patient's immune system could pose greater risks if something goes wrong. For instance, if transplanted immune-stealthy lung cells made from the gene-edited iPSCs acquire a cancer-causing mutation, they could have a higher risk of going on to form lung cancer since the patient's immune system wouldn't be able to detect them. Thus, researchers will have to monitor for such issues during early phase clinical trials.

CRIPSRi-based Genetic Screens for Human iPSC-derived Neurons

The Kampmann Laboratory at UCSF has conducted genetic screens of human iPSC-derived neurons using a CRISPR variant, CRISPRi. CRISPRi, or CRISPR interference, does not lead to the DNA breaks of the original CRISPR-Cas9 system, which can be toxic to iPSCs. Specifically, the nuclease dead Cas9 (dCas9), led by a single-guide RNA, can target points of interest in the genome, and with it researchers can transcription of specific genes in stem cells.

The Kampmann lab used CRISPRi from a , a locus in the genome where genes can be safely inserted and not disrupt other genes, to decrease neuron growth without cutting their DNA. More specifically, desired genes were repressed at different points in neuronal differentiation. The researchers carried out CRISPRi-based genetic screens, which can reveal the gene of interest, its defects that may contribute to brain diseases, and the corresponding cellular pathways that can later be targeted for therapeutic purposes. Researchers also found that "housekeeping" genes, or the genes required to survive and essential for all types of cells, behave differently in neurons and stem cells.

As mentioned in the video below, CRISPRi-modified and iPSC-derived neurons suggest that the genetics of brain diseases can be studied without the of animal models, which often do not entirely accurately portray the biology of the human brain, or donor tissue, which have short-lived neurons.

CRISPR for HIV prevention

September of 2019, researchers at Professor HongKui Deng's laboratory at Peking University published a paper on transplanting CRISPR-Cas9-altered stem cells into a patient with HIV. Specifically, CRISPR was used to modify the genes of donor-derived hematopoietic stem and progenitor cells (HSPCs).

The particular gene of interest was CCR5 because CCR5-null blood cells were shown to be immune to entry of HIV. With the CCR5 mutation as the desired change, Deng's team used CRISPR-Cas9 to genetically modify the CCR5 gene in the HSPCs and transplant these cells into a patient with HIV-1 and acute lymphoblastic leukemia.

However, after the transplantation, the HIV-1 infection of the patient was not fully eliminated. Despite this, there were still promising results from the experiment: the acute lymphoblastic leukemia diminished within , and the CCR5-modified stem cells survived with a CCR5 mutation efficiency of 5.20 to 8.28% . Additionally, there were no harmful effects from the gene-editing process.

Looking Ahead

These recent examples of CRISPR-Cas9 use in stem cells have given insights into disease mechanisms and transplantation biology. There are still challenges with the tool, such as low gene-editing efficiency, off-target binding, low rate of homology directed repair (HDR) compared to nonhomologous end joining (NHEJ), and more. However, CRISPR-Cas9 has become a flexible tool and has been applied to study potential therapeutic methods and targets of various degenerative diseases , including HIV, neurodegenerative diseases, bone/musculoskeletal disorders, cardiovascular disorders, diabetes, and cancer. In addition, CRISPR-related technologies continue to evolve and improve.

Notes

You can see the previous post by the author, Mina Kim, a review of 4 cell therapy types under study for COVID-19 here. Dr. Knoepfler also contributed to this post mainly by brainstorming and editing.

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New research on CRISPR gene-editing in stem cells, infographic - The Niche

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As a pivotal advance in the gene-editing field and timeline, CRISPR continues to be utilized for research on stem cells and human diseases. Today, I will explain some of the most notable recent findings in the stem cell-CRISPR field.

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Learn.Genetics [Internet]. Salt Lake City (UT): Genetic Science Learning Center; 2018 [cited 2023 Nov 27] Available from https://learn.genetics.utah.edu/

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Axolotls: The salamanders that snack on each other (but don't die) - Luis Zambrano

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BIOTECHNOLOGY in the Future: 2050 (Artificial Biology)

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Are GMOs Good or Bad? Genetic Engineering & Our Food

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Biological implications of using PGT testing

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Biological implications of using PGT testing

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Some examples of the biological implications of PGT testing - you need to delve into at least one in depth.

a) Reduce inheritance of specific monogenic disease (PGT-M)

b)Biopsy impacts on embryo viability, mosaic embryo's and impacts on choices if limited embryo's available(single embryo) - False positive/ false negative results.

c) Improving success rates for those couples with advanced maternal age, recurrent implantation failure, and recurrent spontaneous abortions.

d) long term impact on gene pools, genetic variation, etc

e) impacts of thawing process on embryos

f) Reduce risk of having a baby with a chromosomal abnormality (PGT-A)

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Below are links to interactives and videos that cover some of the testing processes;
https://human.biodigital.com/view?id=production/femaleAdult/amniocentesis&amp;lang=e
n

https://www.youtube.com/watch?v=_n3KQNuOCkQ

https://www.youtube.com/watch?v=ImgQyVcC2MU

https://www.youtube.com/watch?v=W4tqLo2iLFU

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Use this source to help guide you.

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The Biology Buddy checklist Bio issue report.docx

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What action could be taken to help with this issue?

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What action could be taken to help with this issue?

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What action could we take;

Governmental level contact to raise issue, petitions with signatures, etc- local MP's, politicians, prime minister,

Protesting - where? What level? When? Where else has this been effective?

Education - Education programs to help with the issue? Where has this worked? Other examples to give you ideas.

Personal changes - What could you do? Small scale changes?

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You must clearly identify at least one opinion in support/for and one opinion against/opposing the biological issue.
These opinions can be from individuals, groups, organisations local or international - try to do local(Aotearoa) if possible.
THEY MUST BE IDENTIFIED!!!
Eg. If it is Joe Bloggs' opinion, then state J. Bloggs when quoting and or describing their opinion.

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The difference between Biological & Societal implications are;
Biological - How does the issue affect living organisms biologically at an individual, population or whole community level.
Societal - How does the issue effect members of society in regards to economics, ethical issues, environmental or cultural aspects.

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Set up a research logbook - digital or hardcopy.

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Set up a research logbook - digital or hardcopy.

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Have a look here for a few tips on starting the research process. The research process.

You might like to consider using google scholar also as a search engine.

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The first step towards writing a research paper is pretty obvious: find sources. Not everything that you find will be good, and those that are good are not always easily found. Having an idea of what you're looking for-what will most help you develop your essay and enforce your thesis-will help guide your process.

Example of a Research Process

A good research process should go through these steps:

1. Decide on the topic.

2. Narrow the topic in order to narrow search parameters.

3. Create a question that your research will address.

4. Generate sub-questions from your main question.

5. Determine what kind of sources are best for your argument.

6. Create a bibliography as you gather and reference sources.

Each of these is described in greater detail below.

Preliminary Research Strategies

A research plan should begin after you can clearly identify the focus of your argument. First, inform yourself about the basics of your topic (Wikipedia and general online searches are great starting points). Be sure you've read all the assigned texts and carefully read the prompt as you gather preliminary information. This stage is sometimes called pre-research.

A broad online search will yield thousands of sources, which no one could be expected to read through. To make it easier on yourself, the next step is to narrow your focus. Think about what kind of position or stance you can take on the topic. What about it strikes you as most interesting? Refer back to the prewriting stage of the writing process, which will come in handy here.

Preliminary Search Tips

1. It is okay to start with Wikipedia as a reference, but do not use it as an official source. Look at the links and references at the bottom of the page for more ideas.

2. Use "Ctrl+F" to find certain words within a webpage in order to jump to the sections of the article that interest you.

3. Use Google Advanced Search to be more specific in your search. You can also use tricks to be more specific within the main Google Search Engine:

1. Use quotation marks to narrow your search from just tanks in WWII to "Tanks in WWII" or "Tanks" in "WWII".

2. Find specific types of websites by adding "site:.gov" or "site:.edu" or "site:.org". You can also search for specific file types like "filetype:.pdf".

4. Click on "Search Tools" under the search bar in Google and select "Any time" to see a list of options for time periods to help limit your search. You can find information just in the past month or year, or even for a custom range.

As you narrow your focus, create a list of questions that you'll need to answer in order to write a good essay on the topic. The research process will help you answer these questions.

Another part of your research plan should include the type of sources you want to gather. Keep track of these sources in a bibliography and jot down notes about the book, article, or document and how it will be useful to your essay. This will save you a lot of time later in the essay process-you'll thank yourself!

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The first step towards writing a research paper is pretty obvious: find sources. Not everything that you find will be good, and those that are good are not always easily found. Having an idea of what you're looking for-what will most help you develop your essay and enforce your thesis-will help guide your process.

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15 Ways to Search Google 96% of People Don’t Know About

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Evaluating Sources - The good, the bad & the ugly

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Evaluating Sources - The good, the bad & the ugly

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The assessment will also be set up in the google classroom as an assignment for your to complete your presentation in.

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A guided view to the tricky biology issues 2022.docx

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Should Aotearoa utilise Pre-implantation genetic diagnosis for assistive reproductive technologies?

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Want to know a bit more about why and how to do this assessment - have a look here.

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Bio3.2 Why Investigate a socio-scientific issue

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To get your head around the idea of socio-scientific issues you first need to know a bit about them.
Complete the task above; Read the quotes and use these ideas as well as your previous knowledge to answer the questions.

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TASK 1 - Socio-Scientific..... but what does that mean?

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Socio-Scientific Research Questions Bio 2026

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Watch 3 different video clips from this playlist, what are the key socio-scientific issues being shown in the videos you watch? What key science ideas need understanding for this issue?
https://youtube.com/playlist?list=PLt844Qbr3s_EibfnXQdToFjKDi0xRV8UA

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Share your videos with friends, family, and the world