Fact Sheet:               SINGLE EMBRYO TRANSFER IS NOT SUITABLE TO BE USED AS CLINICAL INDICATOR FOR PREIMPLANTATION GENETIC DIAGNOSIS

 

Definition of “clinical indicator”: A measure, process, or outcome used to judge a particular clinical situation and indicate whether the care delivered was appropriate.

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In modern human medicine, if any doctor wishes to perform an operation on a patient, the patient should suffer from a disease which will need a surgery to solve the patient’s illness. Without suitable clinical indicator such as how severe or what degree of the disease, the physician will not have any right to perform an operation on the patient, according to recent advanced medicine. Obviously, surgery on every random patient without clinical indicator is illegal in civilized countries.

 

Preimplantation genetic diagnosis (PGD or Preimplantation genetic test, PGT) is the most traumatic or invasive surgery in human medicine, because it is performed at the earliest stage of a life and with about 1/8 of the entity to be removed for diagnoses. Original PGD is performed by two traumatic procedures: intracytoplasmic sperm injection (ICSI) and embryo biopsy at eight-cell embryos. As the thrust of PGD and lack of correct techniques in PGD in these two decades, most of PGD laboratories are not so confident on their single cell amplification techniques. They began to recommend using blastocyst biopsy (or trophectoderm – TE biopsy), which will need five traumatic techniques: ICSI on day 0; assistant hatching on day 3; biopsied embryos; and freeze the biopsied embryos on day 5 waiting for the diagnoses results. In next cycle, the frozen embryos will pass the fifth traumatic technique – thaw the embryos for transferring them to the mother uterus. If these techniques are for prevention of known genetic diseases as the PGD pioneers’ design in 1990s, the benefit is clearly more than harm.

 

Since 2015 ASRM Meeting, some people (Dr. Santiago Munne, Dr. James Grifo, et al.) and some business companies advocated combining Elective - Single Embryo Transfer (eSET) with PGS (preimplantation genetic screening, i.e. PGD not specific for genetic diseases) in human in vitro fertilization (IVF) for every embryo of every patient. The aim is clear and simple: using PGS only for one embryo transfer rather than for prevention of family hereditary diseases. The recommendation of single embryo transfer is correct, and is getting more and more support by the patients and physicians. In a lot of clinics, up to 75 to 80 percent of patients select single embryo transfer according to 2017 ASRM Meeting reports. However, at the same time in 2017 ASRM Meeting, PGS produced a “tornado” of “Mosaicism” discussion. The popular conclusion in this discussion of PGS as a tool for single embryo transfer is as “throw a coin”, for business rather than for science. Safety issue has been surfaced. A lot of patients’ embryos were misdiagnosed as aneuploidy or mosaicism and not to be transferred. Some clinics got up to 10% patients not to be transferred by PGS. Some patients did not believe the said PGS diagnosed results. They insisted to transfer the “aneuploidy or mosaicism” embryos, and produced karyotyping normal healthy babies. However, the patient’s family still worried their child would someday turn to be mosaicism again with psychological suffering in all their life-time. The stories were reported by newspapers including New York Times, questioning correctness of the purpose, techniques, results and billing problems of PGS. PGS has turned as “The Emperor’s New Clothes” for single embryo transfer, as a method for the pseudoscientists to collect money into their pocket and harm the safety of our offspring.

 

SINGLE EMBRYO TRANSFER IS NOT SUITABLE TO BE USED AS CLINICAL INDICATOR FOR PREIMPLANTATION GENETIC DIAGNOSIS. The facts are:

 

First, the recent popular PGD techniques are full of mistakes in every diagnosis step:

A. Most PGD laboratories cannot successfully amplify one copy of one gene 100% with the biopsied cell(s), not to mention amplify multiple genes or chromosome segments 100% correctly. Usually 5 to 7% allele dropout in PCR results are the confirmation1. Seven percent allele dropout means 7% misdiagnoses.

 

B. Now most of the primers for PGS are nonspecific primers (lesser than 20 mers -bases), which will produce misdiagnoses. 2

 

C. Laser biopsy will produce heat, which will coagulate DNA protein. It will greatly influence the DNA to be exposed for PCR amplification. Thus it will significantly lead to misdiagnoses, such as “chaotic” profile and “additional mosaic”, etc.3

 

D. Trophectoderm cells contain a lot of aneuploidy or mosaicism. It cannot represent the inner cell mass which will grow to be a fetus. 3

 

Second, every one of us produce aneuploidy or cancer cells in our bodies all the time. The aneuploidy cells will die or arrest. Immune system will kill the cancer cells. And normal cells will continuously grow. Most of us are not mosaicism. Should we be biopsied all the time in every part of our body?

 

Early human embryos are the same as our body with some aneuploidy cells. Not every cell of the embryos are normal cells. Microtubules in the eggs and early embryos are much longer than later embryo cells. They will be easily broken by changing temperature and by any manipulation or subnormal culture condition. The breakage of those microtubules will lead to some cells turning to be aneuploidy and the embryo turns to be mosaicism. One or two cells of the 8-cell stage embryo are aneuploidy cells will not influence the embryo (with 6 or 7 normal cells) to grow to be normal baby. Five or six aneuploidy cells in the trophectoderm to be diagnosed will not mean the future baby to be aneuploidy or mosaicism. Normal cells are growing more and more, faster and faster, and abnormal cells will die or turn less. Some cells of the embryo are aneuploidy. This does not mean the whole embryo to be aneuploidy. Some cells of the embryo are mosaicism. This also does not mean the whole embryo to be mosaicism, especially considering the misdiagnosis condition with laser biopsy and trophectoderm biopsy techniques. Some normal embryos with part of “aneuploidy cells or mosaicism” is the way of healthy life to be produced. This is a normal physiological phenomenon of our embryos rather than pathological results.

 

If the egg or sperm is chromosomal abnormalities, the zygote will be true aneuploidy. Its future cleavage will produced a lot of fragments due to microtubule and centrosome problems. Most of them will arrest and will not turn to be blastocyst, will not hatch and will not implant in the uterus. Only about 0.1% of them will survive to live birth, mainly Down syndrome, which is the suitable indicator for prenatal diagnoses. When, where, and how often can we see an abnormal live birth child with “chaotic profile3” of abnormal karyotyping?

 

If the zygote is normal, and one cell of the two-cell embryo is normal with another abnormal cell, the abnormal cell will arrest and the embryo will be three-cell embryo next day. It will later grow to be five-cell and seven-cell embryo. If one more cell turns to be abnormal at four-cell stage, the embryo might be six-cell embryo next day, containing four normal cells and two abnormal cells. This is the “aneuploidy and mosaicism” phenomena of the early human embryos. If the culture condition is good, this phenomena will not stop the six-cell embryo which containing two abnormal cells to grow to be a normal baby. More than 99% of our normal babies are produced at this way, normal physiological way. To create “an emperor’s new clothes” - PGS to manipulate our most healthy blastocyst embryos before transfer is misleading in human reproduction.

 

Recent science showed: in human zygotes and early embryos, there are some checkpoint, licence and coordinating proteins, such as Polo and Polo-like kinases 4, ORC2 5 and Geminin6, etc. They are the important “mediators for various cell cycle checkpoints that monitor centrosome duplication, DNA replication, formation of bipolar mitotic spindle, segregation of chromosomes, and mitotic exit, thus protecting cells against genetic instability during cell division.” 4 If the abnormal cells can not pass the checking points to get the licence, DNA of the abnormal cells will not be allowed to be replicated in S phase, and the cells will not be cleaved (i.e cell arrested). These proteins in the zygotes and embryos are similar to our immune system in our bodies, to prevent further happening of the aneuploidy or mosaicism. They are the intra-embryonic natural and effective preimplantation genetic screening (PGS) system. And they are not traumatic. Their functions are much stronger (only 1% live birth to be abnormalities, and true fetal mosaicism to be less than 0.5% 7) than the artificial PGS performing by those pseudoscientists. The artificial PGS will produce a lot of side effects, produce more harm than benefit while comparing embryonic natural PGS.

 

Third, the most important problem is the long term side-effect of popular PGS for every child birth: human beings will be changed to be subhuman beings very quickly in several generations. Before understanding this point, cytoplasm and cytoskeleton function should be reviewed with the most recent scientific research results using the below example – ooplasmic transplantation, which is familiar to many staff working in the field of human reproduction.

 

Children born after ooplasmic transplantation have grown up to 13-18 years old in 2016. Their unhealthy data were published8 and detailed as the following conclusion:

 

“The XO karyotypes were unexpected and were considered potential adverse effects of the procedure8.” Two XO karyotypes were reported out of 14 pregnancies: 14.3%.

 

A quadruplet data (most severe data?) was not published. Another 13 children data were concluded as the following:

 

Neuropathy Problem from 13 children:

Borderline pervasive developmental disorder with attention deficit disorder, depression or suicidal thoughts (male of the twin)                                one

Chronic migraine headaches                              one

Injuries or accidents                                         six/13

Minor vision                                                       three

Attention deficit disorder                                   one

Concern child’s attention span                           three

Concern child’s emotional development            one

Learning disabilities (female of the twin)          one

IQ Problem:

GPA score: Donor’s DNA positive (4 children): mean 3.53, while Donor’s DNA negative (4 children): mean 3.95

(Significantly different: the more donor’s DNA, [i.e. the more cytoplasmic damage], the lower IQ)

 

Immune Problem:

Mild asthma                                                        two

Minor skin problems                                          three

Allergies                                                             Seven/13

Norwalk virus                                                    one, hospitalized

 

Muscle and Other Problem:

Surgery on ear tubes (Donor’s DNA positive)   one

Surgery on shoulder tear                                    one, hospitalized

Minor surgical procedures                                 three

Obesity                                                               one

Borderline underweight at 7th percentile           one

 

Most of the above chromosomal abnormalities, neuropathy problem (IQ problem), immune problems, muscle and other problem are closely related to cytoplasm and cytoskeleton malfunction. The injected cytoplasm contained cytoskeleton and other cytoplasm organelles, which had disturbed the original normal cytoskeleton structure and functions of the recipient oocytes.

 

When microtubules were broken by the injected cytoplasm, it produced aneuploidy (XO karyotyping).

 

After cell differentiation in human beings, the function of cytoskeleton will not only act as (what it is named) cytoskeleton to hold the shape of different kinds of cell structure, but also act as different kinds of tissue or organ functions, such as nerve, muscle and immune system (including heart and sperm) function. When these systems can not function normally, they will produce the above problems. The detailed explanation is outlined as:

 

Cytoskeleton microtubules and actin contain strong polarity. If they are broken and changed their direction, some of them will regenerate to be abnormal structure and they will inherit to next generation9. “Cell polarization generates morphological and functional asymmetry, and is crucial for the development and proper functioning of many cell types.”10 The “microtubule cytoskeleton is a major determinant in the establishment and maintenance of neuronal polarity. Microtubules provide the structural basis for neuronal polarization, because of their intrinsic properties including inherent polarity. Moreover, the polarized microtubule network also forms the basis for selective cargo trafficking into axons and dendrites.”10 “Reduced microtubule stability has been observed in several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS), and tauopathies like Progressive Supranuclear Palsy. Hyperstable microtubules, as seen in Hereditary Spastic Paraplegia (HSP), also lead to neurodegeneration. Therefore, the ratio of stable and dynamic microtubules is likely to be important for neuronal function and perturbation in microtubule dynamics might contribute to disease progression.”11 “The extension of long slender axons is a key process of neuronal circuit formation, both during brain development and regeneration. For this, growth cones at the tips of axons are guided towards their correct target cells by signals. Growth cone behaviour downstream of these signals is implemented by their actin and microtubule cytoskeleton.”12 When the sensor neurons, motor neurons and reflex arc were functioning abnormally, i.e. not coordinating normally, injuries or accidents happened in above six children from total 13 children (46.2%). The other neuropathy problems happened in the 13 children will be easily understood with the above neural change by microtubule perturbation.

 

Normal immune system in our body is related to normal function of cytoskeleton. Normal phagocytosis function of white blood cells will rely on normal structure and normal function of the cytoskeleton to change their greatest angle to finish the action. The “killed target cell would be phagocytosed by neighboring cells (especially macrophages) long before it disintegrated”13. “Striking similarities have been noted between centrosome polarization at the synapse and basal body docking during ciliogenesis”.14 The “centrosome in the TC cell is located at the point of cell-cell contact with the target cell – an immunological synapse. The secretory granules … in the TC cell are initially transported along microtubules to the centrosome, which then moves to the synapse, delivering the granules to where they can release their contents”.13 The “pathogenesis of asthma may be related to the ratio and dysfunction of NKT and CD4(+)NKT cells” in “house dust mite-sensitized children”15. That is why the disorder of cytoskeleton of cytoplasm will produce so many immune problems.

 

Cytoskeleton actin filaments slide past myosin filaments toward the middle of the muscle unit sarcomere, and produce muscle contraction16. Thus disturbance of cytoskeleton will produce muscle and heart problem.

 

Popular PGS for every embryo to be transferred will produce repeated temperature change at least five times, which including freezing and thaw. It is a common sense in the reproductive field that any cooling will produce cytoskeleton damage. To let every embryo get some cytoskeleton to be damaged by temperature change and by invasive techniques before people to get a baby is a severe safety issue which had been confirmed in animal and human reports.17,18 It will produce a lot of children with the above neuropathy17,18, immune and muscle problem. It is a very heavy economic burden to our society in a short time. It is not possible to buffer so great human population of cytoskeleton abnormalities with normal human population of normal cytoskeleton system. When PGS is continuing for several generations as a long time, human beings as a species will be changed to subhuman beings19, full of all kinds of diseases. Change as a new subspecies will be horrified. Cytoskeleton is a very important hereditary materials in our human beings.

 

Fourth, people use PGS to select boy or girl without family history of X-linked diseases, which will disturb natural sex ratio.

 

Base on the above points, Single Embryo Transfer is not suitable to be used as clinical indicator for preimplantation genetic diagnosis. Government should ban the misleading clinical malpractice: using PGS for eSET.

 

A guideline from suitable association for direct transfer one embryo without PGS is appropriate.

 

References:

1. Cui K-H, Haan EA, Matthews CD. Optimal polymerase chain reaction amplification for preimplantation diagnosis in cystic fibrosis (ΔF508).  BMJ 1995, 311: 536-540.

2. Cui K-H, Warnes GM, Jeffrey R, Matthews CD. Sex determination of preimplantation embryos by human testis-determining-gene amplification. Lancet. 1994, 343: 79-82.

3. Sachdev, N.M. et al. Reliability of next generation sequencing (NGS) diagnosis of aneuploidy or mosaicism by re-biopsy and repeat NGS of inner cell mass (ICM) and trophectoderm (TE) from human embryos. Supplement to Fert and Steril, 2017, 108 (35): e87, O-213.

4. Xie, S. Q., et al. Regulation of cell cycle checkpoints by polo-like kinases. Oncogene 2005, 24: 277-286.

5. Prasanth, S. G., et al. Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance. EMBO J 2004, 23: 2651-2663.

6. Tachibana, K. K. and Nigg, E. A. Geminin regulates multiple steps of the chromosome inheritance cycle. Cell Cycle, 2006, 5: 151-154.

7. Capalbo, A., et al. Detecting mosaicism in trophectoderm biopsies: current challenges and future possibilities. Human Reprod 2017, 32: 492-498.

8. Chen, S. H., et al. A limited survey-based uncontrolled follow-up study of children born after ooplasmic transplantation in a single centre. RBO, 2016, 33: 737-744.

9. Livshits, A., et al. Structural inheritance of the actin cytoskeletal organization determines the body axis in regenerating Hydra. Cell Reports, 2017, 18: 1410-1421.

10. van Beuningen, S. F. and Hoogenraad, C. C. Neuronal polarity: remodeling microtubule organization. Curr Opin Neurobiol, 2016, 39: 1-7.

11. Dubey, J., Ratnakaran, N. and Koushika, S. P. Neurodegeneration and microtubule dynamics: death by a thousand cuts. Front Cell Neurosci 2015, 9: 1-15.

12. Prokop, A., et al. Using fly genetics to dissect the cytoskeletal machinery of neurons during axonal growth and maintenance. J Cell Sci 2013, 126: 2331-2341.

13. Alberts, B., et al. Molecular Biology of The Cell. Garland science, Taylor & Francis Group, Sixth Edition, 2015, P. 1334.

14. Stinchcombe, J. C., et al. Mother centriole distal appendages mediate centrosome docking at the immunological synapse and reveal mechanistic parallels with ciliogenesis. Curr Biol 2015, 25: 3239-3244.

15. Yan-ming, L., et al. The effect of specific immunotherapy on natural killer T cells in peripheral blood of house dust mite-sensitized children with asthma. Clin Dev Immunol 2012, 2012: 148262.

16. Cooper, Geoffrey M. (2000).Actin, Myosin, and Cell Movement.The Cell: A Molecular Approach. 2nd edition. Sinauer Associates.

17. Yu, Y. et al. Preimplantation Genetic Diagnosis May Pose Neurological Risks. Mol Cell Proteomics 2009, 8: 1490-1500.

18. Middelburg, K. J., et al. Mental, psychomotor, neurologic, and behavioral outcomes of 2-year-old children born after preimplantation genetic screening: follow-up of a randomized controlled trial. Fertil Steril 2011, 96: 165-169.

19. Cunha-Ferreira, I., Bento, I. and Bettencourt-Dias, M. From zero to many: Control of centriole number in development and disease. Traffic 2009, 10: 482-498.