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Cytohetics: Preimplantation Genetic Screening for Aneuploidy* Is
Not Scientific

* PGT-A is a superstition of Genetics, and it is not according to the biological trait as Genetics described. Thus PGT-A is not a true genetic test but a DNA test without correct genetic results.

Human normal babies are derived from mosaic embryos with normal and aneuploid cells. Most

aneuploid cells in human embryos do not contain hereditary function after helping implantation

and differentiation. The above phenomena can only be explained by Cytohetics.

Ke-Hui Cui M.D., Ph.D.

Savannah, Georgia, 31405, U.S.A.

October 19, 2020

Email: khcui72@hereditics.net

Edited by Dr. YongYan Cui

Abstract

The presence of aneuploid cells in human embryogenesis is an important and beneficial phenomenon of embryonic physiology in implantation and differentiation. Preimplantation genetic screening (PGS) is screening for embryonic aneuploid cells that does not take into account the parent’s history, i.e. biological trait. When “mosaicism” embryos were transferred, they developed into 221 normal karyotype babies. The embryo’s DNA results did not correspond to the baby’s genetic results. In contrast, preimplantation genetic diagnosis (PGD), which accounts for the parent’s genetic history, maintained their embryo’s genetic results to live birth (>99.0%). The positive predictive value between PGS and PGD is significantly different (p<0.0001). Thus, PGS is not a true Genetic test but a DNA test when neglecting the principal component (biological trait) of Genetics. The failure of PGS in hereditary diagnosis is explained by Hereditics (the study of heredity). The patient’s genetic history is closely related to heredity control system (checkpoint and licensing system) in the cytoplasm. And patients’ hereditary diseases result from the combination of defects in both DNA and heredity control system. In PGS, parents without family history of hereditary disease rarely have defects in the hereditary control system. This is why most mosaic embryos will follow their parent’s normal biological trait and produce normal babies.

Keywords: preimplantation genetic screening; preimplantation genetic test; aneuploidy; mosaicism; hereditics; heredity.

History of PGS

In an early case of preimplantation genetic determination (PGD), a male embryo was misdiagnosed to be a female embryo by polymerase chain reaction (PCR) techniques. Thereafter, Dr. Delhanty and Dr, Handyside began to use fluorescent in-situ hybridization (FISH) techniques for sex determination to prevent X-linked hereditary diseases1. With the use of FISH techniques, a lot of human embryos were reported to be aneuploid. In 1997 Dr. Verlinsky claimed that “60% of human embryos are aneuploid”. Is it possible that such a high percentage of embryos are aneuploid when more than 99% of people in the world are normal karyotyping? Severe debate about “aneuploidy” and FISH techniques was initiated in the American Society for Reproductive Medicine (ASRM) meeting in 1997. At the same time, preimplantation genetic screening (PGS) (i.e. preimplantation genetic testing for aneuploidy, PGT-A) was prematurely introduced as a clinical tool for expectant families. The aim of PGS would be: First, “diagnosing and preventing inherited disease” by PGS “may appear useful”2,3. Second, if all embryos obtained by in vitro fertilization (IVF) were screened for aneuploidy before transfer, implantation and pregnancy rates would improve, and miscarriage rates would decrease4. FISH techniques, in which one side of the chromosomes (DNA) adhere to a glass slide and a long-DNA-sequence probe is used, are full of errors. Consequently, array comparative genomic hybridization (aCGH) and next generation sequencing (NGS) replaced FISH techniques. The “aneuploidy” problem is still present in human embryos. After blastocyst biopsy, people started to report, “60% of human embryos have ‘mosaicism”. However, “aneuploidy” and “mosaicism” were myths in the field of human reproduction.

In the 2015 ASRM meeting, Reprogenetics (company) recommended the use of blastocyst biopsy for PGS and elective single embryo transfer (eSET). eSET is highly accepted by society as a means to produce singletons, which is better for the mother’s and child’s health. However, performing PGS involves extremely traumatic techniques including add-on assisted hatching, laser radiation, embryo biopsy, and freezing for every embryo - a majority of which are originally healthy embryos. Given these risks, the combination of PGS and eSET may not ultimately benefit the embryo. Despite this, in 2016, there was a large-scale trend towards performing PGS in the USA. The clinical results in the USA in 2016 did not reflect improved outcomes but instead indicated worsened clinical outcomes with PGS. According to National Summary data from the Centers for Disease Control and Prevention (CDC) in the USA, in the <35 years old group who had embryo transfers, live birth rates were 47.5% in 2014, 46.5% in 2015, and 45.7% in 2016. Miscarriage rates (pregnancy rate minus live birth rate) did not decrease (2014, 7.1%; 2015, 7.3%; 2016, 7.2%)5-7. The second aim of PGS (“implantation and pregnancy rates would improve and miscarriage rates would decrease”) waned when faced with 2015 and 2016 U.S. national data 8.

At the same time, transfer of “mosaicism” embryos produced 221 babies with normal karyotyping9-22. This fact challenged the practice of PGS. How could “mosaicism” embryos confirmed by genetic techniques produce babies with normal rather than mosaic karyotyping? Geneticists failed to answer the question. The PGS-diagnosed aneuploid cells with abnormal DNA structures were not inherited in babies. In the 2019 ASRM meeting, Dr. Rechitsky reported that a total of 800 babies were born in the world after mosaic embryo transfer. Only one baby (i.e. 1/800 = 0.1%) was reported to be chromosomally abnormal23. Thus, there is no difference between the aneuploidy rate in live birth between these PGS-diagnosed aneuploidy embryos and naturally derived embryos (<0.1% mosaicism in fetus)24. With these data, the first aim of PGS (“diagnosing and preventing inherited disease”) was questionable25. The embryo’s genetic results (“mosaicism”) in PGS do not correspond to the baby’s genetic results.

Amidst the turmoil of PGS, the U.S. Congress issued “Personhood” concerns about PGS in March, 2019. It presented four legitimate concerns: First, concern about the concepts of aneuploid embryo and mosaicism embryo. Second, concern about accuracy and applicability of PGS diagnosis. Third, concern about discarded embryos, which is related to ethical questions and humanity. Fourth, concern about safety of PGS. This paper will focus on the first two concerns.

Before discussing the first two concerns, it is necessary to introduce the basic theory of heredity, which is referred to as Hereditics26: Hereditics is the study of heredity. It includes Genetics (study of genes and DNA) and Cytohetics (study of cytoplasmic heredity). The study of expression of genes is known as Epigenetics. The study of expression of cytoplasm, especially in differentiation of cytoplasm, is called “Epicytohetics”.

PGS is not scientific, and it is pointless in heredity

Heredity control system in every human cell

Some cells (especially some dying cells) are aneuploidy. They exist in our bodies, in fetuses, and in embryos and they die continuously. The temporary existence of aneuploid cells is a physiological mosaic phenomenon, rather than a pathological condition – mosaicism27. Cytohetics (study of cytoplasmic heredity) recognizes: Every cell contains a heredity control system, called a checkpoint and licensing system, mainly in its cytoplasm28-34. The checkpoint and licensing system is named for its biological function to control DNA replication, centrosome duplication and spindle assembly. The checkpoint and licensing system is also called the heredity control system when considering its heredity function. It prevents aneuploid cells from passing through the mitotic exit to split and to be inherited in progeny cells32. Thus, the mitotic exit (in the checkpoint and licensing system) functions as a “switch” of heredity in the cytoplasm30. When it is “turned on”, one cell can divide into two cells, and heredity from cell to cell will happen. In animal, cell to cell heredity of an embryo will at last lead to life to life heredity. When the mitotic exit is “turned off” by heredity control system, the aneuploid cell in late embryonic stage will not divide and will then undergo apoptosis, thus ending the said mosaic phenomena35. In contrast, the normal cells continuously pass through the mitotic exit, divide, and grow until an embryo becomes a life - fetus and then a human being without mosaicism. Only when the checkpoint and licensing system of an embryo is defective will the said pathological aneuploid cells in the embryo continuously pass mitotic exit to divide and develop into a baby with aneuploidy or mosaicism27. No self-correction of the defective checkpoint and licensing system was reported, and thus hypothesis of “self-correction” of aneuploidy is questionable27,36,37. The aneuploid cells are degenerated by apoptosis rather than still surviving by "self-correction". An embryo should not be defined as “mosaicism” or “aneuploidy” only based on the presence of aneuploid cell(s) on laboratory results. These cells only represent mosaic phenomena, most of which (799 normal babies/800 babies = 99.9%) is temporary physiological aneuploid cells due to the existing normal heredity control system.

Blastocyst biopsy is not scientific in PGS due to new loose checkpoint and licensing system in fast blastocyst differentiation

The embryo at the 8-cell stage is totipotent. When an embryo grows to the blastocyst stage, the cells have differentiated. Differentiation creates a significant difference between an inner cell mass (ICM) and trophectoderm38. Differentiation also leads to a significant difference in the checkpoint and licensing systems between the ICM and trophectoderm. The ICM, which will later develop into the germ tract, will maintain stricter licensing of transcription and translation39. However, the trophectoderm cells, which will develop into the chorion, will produce new and different loose licensing components40-42. Changes of checkpoint regulator PLK4 were found to be associated with mitotic aneuploidy in 15388 human trophectderm samples. The change of PLK4 are related to the new change of mitotic fidelity43,44. The new loose checkpoint and licensing system allows the trophectoderm cells to differentiate and produce syncytial and aneuploid cells45. In eukaryote (such as in parasite and fungi species), syncytia usually occur with aneuploidy to provide beneficial effects in evolution for adaptation to stress46-49. It is common phenomena that pervasive syncytial and aneuploid cells coexist with normal cells in normal human liver with both beneficial and detrimental consequence50,51. Thus, having many different kinds of aneuploid cells with syncytial cells in the differentiation of trophectoderm is normal phenomenon in evolution and in human physiology. It is not a pathological problem but is normal results from the new loose checkpoint and licensing system in trophectoderm differentiation52. Experimental results showed that 12 embryos biopsied on day 3 were confirmed as normal 2N (i.e. diploid). When they later grew into blastocysts and were analysed as individual cells, they all (except one arrest) contained different kinds of aneuploid cells. This demonstrates that the number of aneuploid cells in blastocysts is significantly higher than that in day 3 embryos due to normal differentiation. In that experiment, 10 of 21 blastocysts contained 4N chromosomes (47.6%) that differentiated to syncytial cells accompanying with all kinds of aneuploid cells53,54. This is a beneficial and benign phenomenon of differentiation in human embryos. Some aneuploid cells coexisting with syncytial cells have a more invasive function for villi to implant into the mother’s endometrium55,56 because “aneuploidy-independent modulation of the microtubule cytoskeleton enhances directional migration” and invasive strength57,58. The aneuploid cells in blastocysts and villi develop into a sharp angular shape, which can be seen under the microscope, and facilitate rapid invasion and implantation into the mother’s endometrium58. Without these aneuploidy cells, implantation will fail and miscarriage will result. The fast invasion allows adequate nutrition to support the fast-growing embryo and fetus, and prevent abnormal functioning of the ICM and embryonic soma. In another study, 42 human blastocysts had their individual cells tested. Almost all embryos (95%) contained some aneuploid cells59. It was assumed that up to 100% embryos contain more or less aneuploid cells60-62. The embryos containing some aneuploid cells are a healthy and physiological phenomenon in the blastocyst stage. These aneuploid cells and syncytial cells in the villi will not result in fetal mosaicism35. In 72,472 pregnancies, chromosomal mosaicism was noted in 2.1% of chorionic villi samples, and <0.1% [(75 – 14) / 72472] in the fetus24. “Overall, the proportion of aneuploid cells is progressively depleted from the blastocyst stage onwards”35. Performing blastocyst biopsy will therefore lead to the finding of more aneuploid cells, and lead to the misdiagnosis of pathological “mosaicism” and unjustifiable discard of normal embryos.

The basic principle of hereditary diseases in Genetics (i.e. patient’s hereditary history) is neglected in PGS

Heredity is the basic principle of hereditary diseases. All geneticists know: in chromosomal abnormalities and genetic diseases, the patient’s history is a valuable source of predicting the probability (such as 25% or 50%) of the offspring’s inheritance of the disease. Patient’s history reveals patient’s biological trait. However, PGS is used to detect aneuploid cells in the general population and does not take into account the patient’s history (or biological trait). In over 800 normal babies born from “mosaicism” embryos, there was only one aneuploid baby23. The PGS positive predictive value (PPV) for aneuploidy was 0.1% (1/800). PGS designers assumed that aneuploid cells would be inherited to offspring because DNA is a hereditary material2,3. However, the majority of aneuploid cells in human embryos are produced by the loose checkpoint and licensing system and are the physiological aneuploid cells, which do not have heredity characteristic40,43,52. This is why PGS repeatedly performed on the same embryos (biopsied on day 3, 5, and 12, or up to stem cells, and up to babies) very rarely have the same results36,37.

Biological inheritance refers to the passing of genetic traits from parents to their offspring. Preimplantation genetic diagnosis (PGD), which is performed based on the patient’s hereditary history, (i.e. following genetic traits) is highly successful. The defective checkpoint and licensing system in these PGD parents has an extremely high likelihood of being inherited to the embryos. Because the inherited defective system is unable to recognize DNA abnormalities, those abnormal embryonic cells will continue dividing and produce babies with the said hereditary diseases27,30. Thus, considering the principle of heredity (based on the patient’s history), the PPV of PGD in diagnosing hereditary diseases in embryos with parents with known hereditary disease is extremely high. In the ESHRE PGD Consortium data from 2011 to 2012, 17,721 PGD cycles resulted in 3,755 cycles with positive heartbeats. Only 34 misdiagnosed cases (0.9%) were reported63. This data supports that PGD has a 99% PPV. It contrasts with a 0.1% PPV when PGS does not take into account the parent’s family history. There are statistically significant differences (chi-squared test p<0.0001) between PGD and PGS in PPV. Based on statistics, if a clinical test cannot obtain a 95% correct diagnosis rate, the clinical diagnosis is insignificant and does not provide any meaningful clinical diagnoses. Thus, PGS is not qualified as a diagnostic test in heredity. Inferred by above theory of Hereditics,  there are two heredity components in Genetics:

PGS-Not Safe 36   Componenets of Genetics 2.png

Genetics alone cannot fully explain the failure of PGS

Genetics accounts for the genes that produce proteins. However, Genetics does not explain how those proteins in the checkpoint and licensing system function as mitotic exit in heredity control system27,30. However, Cytohetics (study of cytoplasmic heredity) explains that mosaic phenomenon will not turn into mosaicism because the checkpoint and licensing system in most embryos (99.9%) is normal rather than defective26. Mosaicism and hereditary diseases result from the combination of defects in both DNA and heredity control system26,27.

Why do most aneuploid cells (99%) in the blastocysts of PGS produce normal karyotyping babies? Many papers described that this phenomenon cannot be explained with our existing knowledge of Genetics. Concerns of false positive results were considered10,11,38,64,65. False positive and false negative results exist at a very low rate in PGS66. It is possible that some artifacts from messy blastocyst biopsy techniques and from the uneven distribution of aneuploid cells can lead to false positives even with advanced molecular technique NGS for PGS14,16,67,68. However, similar genetic test results of PGD with parent’s history has confirmed: it is not possible that any genetic test will produce 99% false positive results, but only about 1% false positive63. A systematical problem in PGS is: neglect of parents’ hereditary history. Accurate DNA results do not translate to an accurate diagnosis of babies that will become (or inherit) aneuploidy24. It was reported that aneuploid embryos diagnosed by NGS also produced 31 normal karyotyping babies19. The advanced NGS genetic diagnosis failed 100% in its heredity prediction in this report was not due to “false positive diagnoses” but due to high efficient functioning of the heredity control system in the aneuploid cells24,35. These findings suggest that genetics and genetic tests cannot fully explain the intricacies of heredity or biological trait in PGS. Genetics is not absolutely equal to heredity under the condition of neglecting family history.

Whether most aneuploid cells contain hereditary characteristics depends on whether their checkpoint and licensing system is normal27,30, and this can be determined based on the patient’s hereditary (family) history (i.e. biological trait). Without considering the patient’s history, PGS becomes a DNA test rather than a true Genetic test of hereditary diagnosis. Thus PGS cannot diagnose aneuploid embryos69. However, when taking the patient’s history into account, PGD becomes a test of both genetic and hereditary diagnosis63. The tool of accurate genetic test is excellent for the object of PGD but is incompetent for the object of PGS. Only recognition of Hereditics (i.e. both Genetics and Cytohetics) and therefore recognition of the importance of patient’s history allows PGT to be a genetic test for hereditary diagnosis. According to four components of Hereditics and two components in Genetics, PGS is not a true Genetic test due to PGS neglecting biological trait of the patient's family from Genetics.

PGS-Not Safe 35   Componenet of Genetics.png

Most aneuploid cells in early embryos have some normal and beneficial cellular functions in human embryos as a result of long evolutionary history

A. Maintaining embryo function. Most aneuploid cells in the embryos may regulate the embryo’s internal environment before undergoing apoptosis. These regulatory mechanisms include maintaining stable temperature, absorbing and transferring nutrition, buffering outside mechanical stress, and maintaining spatial configuration and polarity58.

B. Faster implantation. As mentioned above.

C. Aneuploid cells are related to apoptosis during organ and tissue differentiation. The development of a healthy child requires not only very high rates of proliferation and differentiation, but also apoptosis, which is a crucial mechanism for morphogenesis and the development of the inner organs. Changes in the regulation and rate of apoptosis in more or lesser level have deleterious effects on the trophoblast and consequently the developing embryo and fetus70,71. Without aneuploid cells and without apoptosis, there will be no differentiation, and no healthy human babies. In mammals, cellular proliferation, differentiation, and death accompany early placental development. Programmed cell death is a critical driving force behind organ sculpturing, including mammals, amphibians, and insects as a result of long evolutionary history72. In PGS, 110 human embryos that were biopsied on day 3 and had whole embryo analysis with individual cells on day 5, only one embryo (1%) consistently showed 100% 2N cells. However, that embryo was arrested with 12 cells on day 4 53,62. This confirmed that all growing normal human embryos are mosaic. Without aneuploid cells and apoptosis, the embryo could not grow further. Those rare blastocysts consistently containing 100% 2N cells are not healthy and cannot survive long, due to the lack of function at the time of differentiation.

Possibility of aneuploidy arises from meiotic or mitotic nondisjunction

Aneuploidy arises from meiotic or mitotic nondisjunction73. In meiotic events, it occurred in 5.2% of fresh oocytes74, and 4.5% of human sperm75. In embryos with meiotic nondisjunction, all of the cells in the said embryos will have uniform aneuploidy, which were about 3.9% in all of the analysed embryos (6 identical aneuploid embryos from 154 analysed human embryos)53,54,59. Most of them will arrest in early embryonic stages or will not be able to implant after transfer23. About 96% (100% -3.9%) of human embryos produce aneuploid cells by mitotic nondisjunction59-61. Most of the aneuploid cells in these embryos (99%) contain normal heredity control system and will arrest after blastocyst stage24,35.

Limited representability of aneuploidy rate in PGS diagnosis

In PGS, one cell is biopsied from an 8-cell embryo or 5 cells are biopsied from a blastocyst to represent the aneuploid rate of the whole embryo (which contains about one hundred cells). The faithful representability is about 12.5% (1/8) in a blastomere biopsy or 5% (5/100) in a blastocyst biopsy76. If FISH has a correct diagnosis rate of 95% in the biopsied cells77, the faithful representability of the biopsied cells in the whole blastocyst is 4.75% (0.05X0.95). If NGS is 100% sensitive67, the faithful representability is 5.0% (0.05X1.00). Thus, the improvement of genetic techniques68 does not greatly improve the representability of aneuploidy rate in PGS diagnosis.

When embryos have undergone repeated biopsy and testing, PGS showed discordant results38. The reason may be: different aneuploid cells in the same embryo are distributed unevenly in different locations11,65, or the heredity control system changes to looser or stricter condition as mentioned above. This is a problem of representability of the biopsied cells. Five-cell results cannot represent the hereditary condition of the whole embryo, or the embryo’s genetic results do not represent the baby’s genetic results. Thus the diagnosis of “euploid”, “mosaic” and “aneuploid” based on a percentage of aneuploid cells found by PGS12,68,78 is subjective.

 

Falsification of the “advantage” of PGS

The “advantage” of PGS has be focused for a long time in the second aim of PGS: “implantation and pregnancy rates would improve, and miscarriage rates would decrease”. Many prominent papers have shown the “advantage” of PGS, which provided an opportunity to be studied once more:

In advanced maternal age (AMA), oocyte nondisjunction increases significantly74. Thus, AMA was the first target of PGS. A report showed that the PGS group had a 37% pregnancy rate and 22.5% implantation rate while the control group yielded 27% pregnancy rate and 10.2% implantation rate79. However, the conclusion that PGS “has immediate impact on the ongoing implantation rate, especially in patients aged ≥38 years” was premature. It was contradicted by its later description: “The pregnancy rate per started cycles does not differ between the study and the control groups”. And PGS “leads to a statistically significant reduction in the number of cycles transferred (78% versus 93% in the controls; P<.001)” 79. Another study reported: “More than 3000 clinical cycles indicates the positive impact” of PGS for AMA patients80. Two years later, the same author, Dr. Verlinsky reported contrary conclusion: in older women, PGS “has a high cancellation rate and low cycle outcome”81. More papers confirmed that PGS significantly reduced the rates of ongoing pregnancies and live births in women of AMA82-84.

Another misperception of PGS stems from the confusion between PGS and PGD. In a study of “PGD cycles”, using the results of 7.8% (183/2359) PGD cycles to represent the results of 92.2% (2176/2359) PGS cycles was inappropriate85. The take-home baby rate after PGD was 23% (42/183) per cycle rather than 81.4% as reported in the abstract85. Using the advantage of PGD to describe PGS, or mixing up the PGD and PGS results86 is confusing in science.

Studies comparing the PGS data with general frozen embryo transfer (FET) data (as a control group)87-89 are also not convincing. The general FET embryos are the leftover embryos after fresh transfers, and therefore of lower quality and not suitable to be used as a control for comparison.

The most common method is to compare the PGS group with the control group by “per transfer” rather than by “per cycle”. No embryo transfer (ET) is quite usual in PGS. “No ET” rates can vary significantly: 17.2%90, 20.7%91, 25% (2003 CDC data)92, 31.0%93, 37.4%94, 52.8%95. High transfer cancellation rates correlated with reduced live-birth rates96. The patients per cycle is the total number of patients with intention to treat (ITT), while patients per transfer is a selected group within the whole group. Only using a selected group to show the advantage in the whole group receiving treatment is misleading. On the whole, when PGS was compared “versus morphology as selection criteria” for embryo transfer, PGS “did not improve overall pregnancy outcomes in all women, as analyzed per embryo transfer or per ITT”8,97,98.

Conclusion

PGS (PGT-A) is a large-scale practice that does not follow the principle of heredity and Genetics (i.e. the patient’s hereditary history). It screens aneuploid cell(s) at the embryonic differentiation stage in an attempt to diagnose hereditary diseases. It operates under the assumptions that: 1. the DNA of aneuploid cells always contain hereditary characteristics that lead to abnormal babies; 2. aneuploid cells only harm rather than help implantation; and 3. only embryos containing 100% euploid cells (normal DNA) are the best embryos. These assumptions are incorrect according to the above review. PGS is a type of misunderstood “Genetics” that lacks scientific basis in embryo differentiation and evolution. It is based on the wrong theory that DNA is the unique hereditary material in human beings, and neglects hereditary function of cytoplasm which is called the checkpoint and licensing system. Thus PGS is a kind of DNA tests rather than a true Genetic test. However, taking into account the patient’s history, PGD is not only a test of genetic diagnosis but also a test of hereditary diagnosis. In PGS, while human “mosaicism” embryos are transferred, they follow their parent’s biological trait and develop to normal karyotyping babies in a rate of 99.9%.

 

 

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