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

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

March 15, 2019

Email: khcui72@hereditics.net

Edited by Dr. YongYan Cui

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​Human beings have evolved over millions of years and maintained the heredity of both genome and cytoplasm. Now, we are facing the challenges of 1) overpopulation which can lead to damage of the external environment and 2) artificial techniques that disturb the stability of natural cytoplasm and genes in the human germline. Both of these challenges threaten normal and natural human heredity. The origin of the (cytoplasmic) heredity has been traced to the beginning of eukaryotes and sexual reproduction, last universal common ancestor (LUCA), RNA world, and to the origin of life (i.e. a combination of RNA and lipid membrane). Mitochondrial replacement techniques (MRT) disturb hereditary materials –such as cytoskeletons and the heredity control system (checkpoints and licensing system). It affects differentiation of different organ systems and produces subhuman beings with inferior species quality. MRT is not scientific or natural in human heredity. The study of heredity should include studies of both genes (DNA) and cytoplasm.

* This was one of two attached papers in Dr. Ke-Hui Cui's Letters to FDA (U.S. Food and Drug Administration), CDC (Centers for Disease Control and Prevention) and ASRM (American Society for Reproductive Medicine) on March 15th, 2019.  Another attached paper was "Fact Sheet: Preimplantation genetic test for aneuploidy (PGT-A or PGS) is lack of scientific bases and is not safe".

The evolution of human beings

All great apes apart from human have 24 pairs of chromosomes [Grouchy, 1987]. There is a strong hypothesis that the fusion of two of shorter ancestors’ chromosomes created a longer human chromosome 2. Thus human have 23 pairs of chromosomes [MacAndrew, 2006; Yunis, et al. 1980; IJdo, et al., 1991]. Since the human-chimpanzee divergence in genomics, human beings have existed on our earth for 4.6 to 6.2 million years according to molecular clock [Chen and Li, 2002]. Over a long period, human beings stood up, freed their two hands, performed labor, communicated together, educated offspring, and at last became distinguished from animals [Gould, 1994].

Since then, human reproduction was in relatively stable condition in quantity and quality during the majority of the last 5 million years. The quantity of human reproduction is mainly expressed as human population. The quality of human reproduction is mainly expressed as heredity of humans. Nevertheless, human beings are now facing a new milestone in both quantity and quality.

The Scientific and Industrial Revolutions and population growth

Genetic information suggests that around 70,000 years ago there was a dramatic collapse in the world human population, falling to very low numbers (10,000 -30,000 individuals), due to the eruption of the Toba supervolcano in Indonesia [Dawkins, 2004; Hawks, et al. 2000]. Subsequently, numbers recovered, and the development of agriculture and more settled communities enabled populations to regrow. Despite localized population collapses brought about by famine, war, and disease, the population continued to slowly increase on a global scale, reaching between 200 and 300 million by 1 AD. It then took the next 1,600 years to double to 600 million in 1600 and reached around 800 million by 1750s [UN, 1999]. The Scientific Revolution in 17th and 18th centuries was characterized by the emergence of modern science, including developments in mathematics, physics, chemistry, astronomy, biology, and human anatomy [Galilei, 1974; Moody, 1951; Clagett, 1961; Maier, 1982; Hannam, 2011; Grant, 1996]. The leading figure of the Scientific Revolution was Isaac Newton and his 1687 “Principia”, which encompassed the laws of motion and universal gravitation [Thomson, 1786]. The Scientific Revolution was the prologue and cornerstone of the coming Industrial Revolution. Human population increased significantly when the Industrial Revolution was underway from the 1760s, with invention of the textile machine, steam engines, and more machine tools [McKeown, 1976; Landes, 1969, P.10 and P.82; Mingay, 1986]. Industry, agriculture, and science developed faster and faster, especially in the last 70 years. Penicillin was discovered on the morning of Friday September 28, 1928 by Scottish scientist Alexander Fleming and, by 1942, it was used to treat infections [Derderian, 2007; Torok, et al. 2009]. Antibiotics and other scientific developments greatly improved the infant survival rate and extended human life expectancy. The advanced effect of antibiotics is the most obvious and important factor for human population growth when compared to other industrial and agricultural factors. The human population suddenly grew from 2 billion in the 1950s to over 7 billion presently [Gerland, 2014]. It is almost four times the population it was 70 years ago and is still increasing [Population Reference Bureau, 2013].

People prefer natural and “green”

In the last century, artificial fertilizers [Smil, 2004] and insecticides [Stephenson, et al. 2006] have enabled us to produce enough food to eat; artificial fibers have provided us with sufficient clothes to wear; and cars are efficient tools for transportation. Initially, “artificial” was a positive term and a strong trend. However, a lot of negativity about the word “artificial” soon became visible in our daily life: The air was polluted by industrialization, farming fields were hardened by artificial fertilizer, water and food were contaminated by insecticides, and men’s sperm counts decreased by 50-60% within four decades due to a variety of artificial chemical substances [Levine, et al. 2017]. These incidences showed that the earth has limited resources [Brown 2012]. And it has exceeded its limits for natural recirculation and cleansing of the air and water, and exceeded its limits of normal temperature and resources of ores, etc. “We want green (natural environment)” has unexpectedly become a popular and positive slogan in our world in the last 15 years, opposite the views of the prior 300 years. The word “artificial” has lost its luster, now adopting a negative connotation in our environment. The old mentality has been replaced by the desire for a more “natural”, “organic”, or “green” outlook in both our daily lives and in some aspects of reproductive medicine.

The birth of cloned sheep, Dolly, [Wilmut et al., 1997] was the apex of the “artificial” era in reproductive medicine. What followed was the birth of infants by mitochondrial replacement techniques (MRT) in human reproduction via ooplasm transfer [Cohen et al. 1997]. People applauded this. However, the trend in the world quickly shifted. As a result of incomplete reprogramming, Dolly developed a lot of health issues including immunological problems. At the same time, the implications of MRT were exposed as it was revealed that human genes had been changed in this process [Barritt, et al. 2001]. Criticism towards MRT began, but there was immediate pushback. The criticism of MRT was dismissed as “negative” in mainstream of reproductive medicine in 1999. MRT was still publicly thought to be “a science” even up to 2016 because DNA (genes) was thought of as the only hereditary material in the world, and ooplasm transfer would not change gene expression. No one at that time thought of cytoplasm as hereditary material. However, people have begun to realize the negative effect that MRT changes the cytoplasm and the heredity of human beings, and that it has crossed a redline of normal and natural human genetics, which would inevitably change our offspring forever [Cui 1999].

Elucidating how the cytoplasm is related to heredity, differentiation, and life evolution is very difficult [Cui 1999]. The characteristics of cytoplasmic heredity can be traced step by step, starting from the basis of cell anatomy, organ and tissue differentiation, special inheritance patterns of cytoplasm, heredity control function of centrosome in the cytoplasm, differentiation function of centrioles in the cytoplasm, eukaryote evolution and centrioles, sexual reproduction, last universal common ancestor, and RNA world. When tracing the origin of heredity and the origin of life, RNA and lipid membrane were earliest predecessors, rather than DNA. Thus, it confirmed that the cytoplasm was earlier than DNA in obtaining heredity function. All of these tracing steps (i.e. confirmation courses) are detailed after the description of overpopulation and its control.

Overpopulation and its future control

Protecting our environment in terms of pollution and contamination is a compelling task for the world leaders. However, human overpopulation is the most pressing factor of environmental pollution and is neglected by most countries [Ehrlich & Ehrlich, 2004]. According to the Merriam-Webster dictionary, overpopulation is “the condition of having a population so dense as to cause environmental deterioration, an impaired quality of life, or a population crash”. By now, most people in the world are aware that the environment is deteriorating. People in first world countries can detect a change in quality of life. And some countries such as China are threatened by a population crash, prompting the change of “one family one child” into “one family two children” policies to prevent this. As the world population grows, the side effects of overpopulation will influence all countries to change their policies in politics, economics, and health. The reasoning behind this is: as the population of each country grows, there will be a lower ratio of available resources per capita, resulting in a higher inflation rate and harder life in the said country [UN 2001]. Additionally, with a growing world population, there will be fewer areas covered by trees and forest, resulting in worse weather and less natural water production. With all of these diminishing resources, the world will be changed into an unrecognizable condition. “The greater population, the better” is a past tense ideal in most countries nowadays. “A suitable population with better quality offspring in health and education” will be the aim of future human beings.

Between 1950 and 2005, the number of children born per woman in the world decreased from 5.02 to 2.65 [UN 2011]. In spite of that, world population stabilization was foreseen as unlikely in this century [Gerland, et al., 2014]. Decreasing the human population growth rate to zero, (i.e. to the plateau of population) by 2050, and then to even further slow it down to be slightly negative (i.e. the population becoming smaller), is the hardest and most urgent task for human beings [UN 2011].  It is also one of the most basic and effective measures for environmental control. To what degree will the human population be controlled? This is a new research subject for scientists, as consumption by human beings should be balanced with the carrying capacity of the earth [Ewing, et al. 2009].

QUALITY OF HUMAN REPRODUCTION

Although the quantity of human reproduction is increasing, the quality of human reproduction has been decreasing. In the last several decades, there has been an obvious decline in sperm count [WHO 1999, Levine, et al. 2017]. In addition to stress, which can influence sperm count [Cui 1996], environmental factors including pollution are linked to epigenetic alterations of male infertility [Kitamura, et al. 2015]. These alterations are also reflected in the recent increasing number of oocytes found with severe abnormal morphology in in vitro fertilization (IVF). Accompanied with environmental factors that have changed the quality of human reproduction, some agricultural techniques (originating from animal research) and other new artificial techniques have also been introduced into human reproduction to change the quality of human reproduction in the last twenty years.

Concepts of cloning and pronuclear transfer and their ban

Mitochondrial replacement techniques (MRT) include both pronuclear transfer (PNT) and spindle transfer. In trials of pig PNT, forty-nine stillbirths were reported [Prather, et al. 1989]. Many stillbirths were also reported in mice PNT and spindle transfer research [Liu, et al. 2003]. PNT in human embryos was trialed in 2003 with the aim of improving human reproduction and preventing transmission of mitochondrial DNA diseases, but these also resulted in stillbirth [Zhang, et al. 2003]. Reporters have criticized these techniques as cloning [Regalado, 2003; Boseley and Watts, 2003]. However, the difference between cloning and PNT should be made clear. Cloning (i.e. somatic cell nuclear transfer - SCNT) uses a differentiated cell to copy the cell provider, while PNT uses a non-differentiated cell, i.e. totipotent cell, to produce a brand new life with three parents’ hereditary materials. While cloning, PNT, and spindle transfer all include genome transfer, only cloning and PNT include nuclear transfer. In cloning, the differentiated cell is changed to a totipotent cell via reprogramming. If reprogramming was performed as perfectly as meiosis is, most of the expressed gene DNA would need to perfectly undergo complicated chemical reactions such as methylation. However, cloning techniques were not successful enough to achieve this [Cui, 2005]. The success rate of cloning (SCNT) in many kinds of animals is very low and only 1 – 3% of reconstructed embryos develop to term. In cases of live birth, some cloned animals are abnormal and die prematurely [Fulka and Mrazek, 2004].

In 2001, the U.S. House Referendum passed the Human Cloning Prohibition Act (H.R. 2505), and the Food and Drug Administration (FDA) imposed regulations to restrict MRT with ooplasm transfer to produce human offspring [FDA 1999; FDA 2002]. In China, the Department of Health banned PNT and gene transfer for human reproduction in 2003 [Grady, 2003].

First birth of artificial babies via spindle transfer

While cloning is banned in humans, research enthusiasm for human MRT has slowly increased under restricted conditions in the last twenty years [Craven, et al. 2010; Tachibana, et al. 2013; Kang, et al. 2016; Hyslop, et al. 2016]. The Mitochondrial Augment Program by OvaSciences suggested that artificially extracted mitochondria should be injected into every egg to produce our human babies [Cui, 2017a]. That was part of an international cohort of MRT supporters with a symphony of public media [Johnson, 2016]. Accompanying this very strong background, live birth of the first baby of this kind of artificial subhuman being (in about 5 million years of human history) produced by spindle transfer was reported by Dr. John Zhang in 2016 in the U.S. [Zhang, et al. 2017]. While this birth was a living phenomenon (similar to the cloned sheep Dolly), it cannot be used to confirm the safety of the spindle transfer technique in a statistically significant manner. Despite claims that MRT does not change epigenetics of the MRT embryos and that MRT babies contain normal karyotyping by Preimplantation Genetic Diagnosis (PGD), Dr. Zhang’s work and production of MRT babies was very bad news for human beings, patient families, and for true scientists. Why? Because only focusing on a small part of the cell’s biological function of the mitochondria and neglecting the whole cytoplasmic hereditary function of the cells is not scientific. The basic sciences of cellular anatomy and molecular biology are important tools for understanding how MRT changes heredity, as itemized and discussed in the next sections. The superior hereditary genes, chromosomes, microtubules, and mitochondria in the original eggs, sperm, and embryos (i.e. in the germline cells) are disturbed, torn, mixed, and changed during MRT. They become inferior, artificial and abnormal genes, chromosomes, microtubules, and mitochondria in an abnormal cellular atmosphere with abnormal structures, contents, and locations. In this way, MRT causes human reproduction to become subhuman reproduction [Cui, 2017b]. SCNT, PNT, and spindle transfer techniques have been shown to have very detrimental effects on eggs, zygotes, and on their future development - from low embryo cleavage rate, low blastocyst rate, high miscarriage rate to high stillbirth rate and all kinds of after-birth abnormalities and early death [McGrath and Solter, 1983]. The FDA banned MRT again in 2017 [FDA, 2017].

What is the reason for the low success rate in SCNT and MRT (PNT and spindle transfer)? In SCNT, the deficiencies in genome reprogramming cause problems in DNA methylation and histone modification, which are important for producing healthy embryos [Fulka, et al. 2001; Martin, et al 2006; Egli, et al. 2011; Noggle, et al. 2011; Mason, et al. 2012]. Apart from the problem of reprogramming in SCNT specifically, SCNT and MRT both damage normal cell anatomy of oocytes and zygotes. In cell anatomy, the most basic cell structure is the cytoskeleton. “The cytoskeleton’s varied functions depend on the behavior of three families of protein filaments – actin filaments, microtubules, and intermediate filaments. Each type of filament has distinct mechanical properties, dynamics, and biological roles, but all share certain fundamental features. Just as we require our ligaments, bones, and muscles to work together, so all three cytoskeletal filament systems must normally function collectively to give a cell its strength, its shape, and its ability to move.” [Alberts, et al. 2015 P.889]. More complicated differentiation and heredity functions of the cytoskeleton were unearthed in recent 10 years.

Normal cell anatomy and biology

Microtubules are always associated and distributed with actin, endoplasmic reticulum, and mitochondria together [Soltys and Gupta, 1992; Shin, et al. 2004] for structural stability [Sui and Downing, 2017]. One end of the microtubule is connected to dynein which attaches to the cell membrane, and this end of the microtubule also connects to dynactin which further attaches to actin-related protein [Alberts, et al. 2015 P.939]. The other end of the microtubule connects to a centrosome and is further “linked across the nuclear envelope to the nuclear lamina or chromosomes” [Alberts, et al. 2015 P949]. Thus, all chromosomes in eggs and zygotes are not free floating in the nucleoplasm and cytoplasm. They are anchored to the nuclear membrane and then further anchored to the cell membrane by microtubules and endoplasmic reticulum, actin, etc. in very specific order. Although chromatin can move to specific sites within the nucleus to alter gene expression, “individual chromosomes have shown that each of the 46 interphase chromosomes in a human cell tends to occupy its own discrete territory within the nucleus” [Alberts, et al. 2015 P211]. “Gene-dense chromosomes localize to the interior of the nucleus, whereas gene-poor chromosomes are located more peripherally”. [Martins, et al., 2012]. The centrosome “is located near the nucleus and from which microtubules are nucleated at their minus ends, so the plus ends point outward and continuously grow and shrink, probing the entire three-dimensional volume of the cell”. “Embedded in the centrosome are the centrioles,” “where microtubule nucleation takes place” [Alberts, et al. 2015 P930]. The microtubules connecting from the centrosome to dynein at the cell cortex are called astral microtubules. Oscillatory nuclear movement allows migration of chromosomes during meiosis and “is mediated by dynamic instability and selective stabilization of astral microtubules” [Ding, et al. 1998]. The microtubules connecting from centrosome to kinetochore of centromere region of the chromosomes are called kinetochore microtubules. The spindles use them to pull the chromosomes apart during prometaphase and metaphase with microtubule flux, while interpolar astral microtubules push the arms of the chromosomes away from the spindle poles to “help align the sister-chromatid pairs at the metaphase plate [Alberts, et al. 2015 P991]. Thus, the specificity in the location of chromosomes and their associated microtubules are important for normal cell division.

Cytoplasm disarray created by SCNT and MRT

During SCNT and MRT, the genome complexes of cells are extracted out of the donor cell and placed into the recipient cell. In the donor cell, the nucleus holding the genome complex is originally connected by microtubules to the cytoskeleton. As the nucleus and its genome complex are extracted, all of the cytoskeleton that is originally connected to the nucleus and the genome structure breaks [Zhang, et al. 1999; Tachibana, et al. 2013; Bai, et al. 2006]. The ends of the broken cytoskeleton are not cleanly cut but instead are fragmented with small amounts of cytoplasm, due to shear force [Tachibana, et al. 2013]. The broken cytoskeleton and integrins greatly disturbed the stability of nuclear structure and produce all kinds of chaotic phenomena [Maniotis, et al. 1997].

Cytoskeletal actin and microtubules contain inherited polarity. In excision of Hydra (the best cytoskeletal experimental model), axis direction of actin in excised pieces determined whether the regenerating offspring would be normal (single body axis) or abnormal (multiple body axes). When the excised axis direction was abnormal, about two-thirds of offspring also showed abnormal body axes or multiple axes. Even when the excised axis direction was correct, about 10% of offspring still showed abnormal body axes or multiple axes [Livshits, et al. 2017]. Microtubules are the same. In Xenopus (frog) eggs, when microtubules were severed, they would “depolymerize from their newly exposed plus ends” [Tirnauer et al. 2004]. This can explain why, in bovine oocytes that underwent chemically induced enucleation, “approximately 50% of treated oocytes presented microtubule reduction” [Saraiva, et al. 2015]. Basic experiments showed that broken microtubules cannot recover as their original selves. Instead, microtubule breakage leads to shorter astral fibers with abnormal microfilamental pattern and abnormal distribution, along with abnormal actin distribution [Spurck, et al. 1990; Zhu, et al. 2007; Yoo, et al., 2007; Kwon, et al., 2010; Fan, et al. 2009; Poehland, et al., 2008]. In Tirnauer et al.’s research, “larger spindle fragments contained a higher percentage of stable microtubules” that could maintain their position after breaking [Tirnauer et al. 2004]. However, other experiments have shown that “after the arm of a …cell is cut off with a needle, microtubules in the detached cell fragment reorganize so that their minus ends … buried in a new microtubule-organizing center,” or centrosome [Alberts, et al. 2015 P932]. Thus, the old microtubules that have separated will be difficult to reconnect at the same broken point again. At the same time, multiple microtubule-organizing centers (MTOCs) are produced instead of the normal, original one or two MTOCs. They worked as multiple centrosomes “resulting in multiple spindles” [Paull, et al. 2013; Egli, et al. 2011; Balbach, et al. 2007]. In SCNT of bovine oocytes, “abnormalities in either distribution and/or number of centrosomes were evident in approximately 50% of reconstructed embryos” [Dai, et al. 2006]. Because “alterations of centrosomes are linked to aberrant cell cycle progression, aneuploidy, and tumorigenesis in many cell types,” “spindle defects resulting from centrosome and motor deficiencies … produce aneuploidy preimplantation embryos, among other anomalies including genomic imprinting, mitochondrial and cytoplasmic heterogeneities, cell cycle asynchronies, and improper nuclear reprogramming” [Simerly, et al. 2004]. Centrosome abnormalities resulting from MRT can also have catastrophic consequences. This is evidenced in bovine oocytes after SCNT, the “oocytes have aberrant spindle morphology and SAC (spindle assembly checkpoint) … may be responsible for chromosome instability” [Tani, et al. 2007]. Furthermore, MRT has been reported to produce epigenetic alterations resulting in lower levels of gene expression [Paull, et al., 2013].

The quoted references above were specifically selected from basic research of cellular anatomy and cellular biology to explain why there are multitude of papers published about the vast arrays of the abnormalities in offspring after SCNT and MRT. According the evidence above, the pathology related to the abnormal cellular anatomy in MRT and its creation of a subhuman species [Cui, 2017c] is concluded as:

A. LACK OF RECONNECTION BETWEEN MICROTUBULES AND CHROMOSOME.

B. ABNORMAL LENGTH OF MICROTUBULES.

C. CROSSOVER OR TWIST OF NEW CELL (ZYGOTE) MICROTUBULES AND CHROMOSOMES.

D. DISLOCATION OF CHROMOSOMES.

E. DISCONNECTION OF NORMAL COMMUNCATION IN CELLULAR ORGANELLES.

F. MULTIPLE CENTROSOMES.

G. ABNORMAL SPINDLE CHECKPOINTS.

The points of D, E, F, and G are closely related to oocyte polarity. The normal oocyte polarity includes asymmetries in duplication of centrioles and formation of centrosomes [Clapp and Marlow, 2017; Heim, et al. 2013]. Oocyte polarization is coupled to the chromosomal bouquet, or chromosome territories, a conserved polarized nuclear configuration in meiosis. That is, the polarity of the oocytoplasm is coupled to the polarity of the chromosome territories in three-dimensional spatial configuration [Elkouby, et al. 2016]. The polar chromosome territories align to build up higher order compartments in specific orientation of replication, and this organizational principle is inherited in human beings [Sadoni, et al. 1999] through cell division [Bornens, 2008]. SCNT and MRT damage the inherited polarity alignment between the cytoplasm and the nucleus, or genome, by extracting one genome and inserting another genome. SCNT and MRT trigger “profound chromatin rearrangements including the dispersion of the donor cell chromocenters components” [Martin, et al. 2006].

Can the reconstructed oocytes or embryos be 100% identical to their original, natural components after SCNT and MRT? The scientific evidence above proves that this is not possible. The microtubules and actin filaments that are reconstructed by SCNT and MRT are not the same as our natural, inherited ones that have been passed down over five million years of evolution of human beings. The damage of microtubules and actin by MRT also influences the MRT oocytes or zygotes to differentiate into abnormal neuron cells, muscle cells, and other cells in different tissues and organs as evidenced below.

Cytoskeleton and neuron differentiation

“Recent data have revealed that 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” [Van Beuningen and Hoogenraad. 2016]. The differentiation of nervous system starts with the growth cone of an axon. “Navigation of the growth cone at the tip of the developing axon is crucial for the proper wiring of the nervous system” [Kahn and Baas, 2016]. The growth cone is a fan-shaped structure that has two domains: central domain (microtubule-rich region) and peripheral domain (actin-rich lamellar region) [Dent and Gertler, 2003]. The motility of the growth cone (during axon extension, retraction, and turning) is achieved by actin protrusion first, and then facilitated by microtubules [Gallo, 2011]. And “severing in the labile portion of the microtubule affects the capacity of microtubules to assemble labile portions into the peripheral domain of the growth cone” [Kahn and Baas, 2016; Prokop, et al. 2013]. In addition to the growth cone, axon collateral branches are important during neuron differentiation, as they allow individual neurons to make contact with multiple neurons within a target and with multiple targets [Gallo, 2011; Delandre, et al. 2016]. The formation of axon collateral branches is reliant on the microtubule-associated and severing proteins.

“As they (neurons) differentiate, neurons send out specialized processes that will either receive electrical signals (dendrites) or transmit electrical signals (axons). … Both axons and dendrites … are filled with bundles of microtubules that are critical to both their structure and their function. In axons, all the microtubules are oriented in the same direction, with their minus ends pointing back toward the body and their plus ends pointing toward the axon terminals. The microtubules do not reach from the cell body all the way to the axon terminals; each is typically only a few micrometers in length, but large numbers are staggered in an overlapping array. These aligned microtubule tracks act as a highway to transport specific proteins, protein-containing vesicles, and mRNAs to the axon terminals, where synapses are constructed and maintained. The longest axon in human body reaches from the base of the spinal cord to the foot and is up to a meter in length. … The microtubules in dendrites lie parallel to one another but their polarities are mixed, with some pointing their plus ends toward the dendrite tip, while others point back toward the cell body” [Alberts, et al. 2015 P.940-941]. Due to the specific positional alignment of over millions and millions of microtubules in natural intact neurons, our bodies are able to sense and react to stimuli precisely without mistakes. Because SCNT and MRT greatly disturb the natural order of the microtubules, neurons differentiated from the millions of impaired microtubules will exist with wrong location and wrong direction, and will not function normally. They will function inferiorly or will have complete absence of function. “Within a neuronal cell, microtubules (MT) are found to have variable lengths and can be both stable and dynamic (or turnover, i.e. dynamic exchange between MT polymers and free tubulin dimers). … 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” [Dubey, et al. 2015]. When microtubules are abnormal, the increased number of dynamic microtubules will lead to increased neuronal branching, shortened neutrites, axons that do not connect to postsynaptic cells, and impaired synapse functioning [Figure 2 of Dubey, et al. 2015]. Experiments show that “hyperdynamic microtubules impair axonal transport and accelerate motor neuron degeneration” “but not in sensory nerves” [Fanara, et al. 2007]. This compromises the coordination and interaction between the motor and sensory nerves, which ultimately predisposes to more injuries or accidents. It was reported that six of thirteen children born after ooplasmic transplantation [Cohen, et al. 1997] had history of injuries or accidents [Chen, et al. 2016]. The report confirms that the injected ooplasm containing donor microtubules, actin, and other cellular organelles disturbed the cytoskeletons of recipient oocytes. These abnormal microtubules could not return to their normal length, numbers, or spatial positions in the recipient oocytes, and such abnormalities were inherited to differentiated cells. Thus, they produced abnormal neuron function [Pessoa-Pureur and Wainer, 2007], one of the symptoms of MRT syndrome. Many different neuropathic problems were reported in the 13 children. In these children, the mean GPA of donor’s DNA positive (4 children) was 3.53, while the mean GPA of donor’s DNA negative (4 children) was 3.95.

Cytoskeleton and immune system differentiation

Normal cytoskeletons of germline cells are not only important for normal differentiation of the nervous system, but also important for differentiation of other systems. In the immune system, “many cells require rapid cytoskeletal rearrangements for their normal functioning during interphase as well. For example, the neutrophil, a type of white blood cell, chases and engulfs bacterial and fungal cells … by extending a protrusive structure filled with newly polymerized actin filaments. When the elusive bacterial prey moves in a different direction, the neutrophil is poised to reorganize its polarized protrusive structures within seconds” [Alberts, et al. 2015 P.890-892]. Normal functioning of macrophages and lymphocytes (cytotoxic cells or natural killer cells) also depend on the intact cytoskeletal structures [Alberts, et al. 2015 P.1334]. “Cytotoxic T lymphocytes (CTLs) are highly effective serial killers capable of destroying virally infected and cancerous targets by polarized release from secretory lysosomes … focusing microtubule-directed release at this point” [Stinchcombe, et al. 2015]. “An increasing number of studies have revealed associations between pre- and perinatal immune activation and the development of schizophrenia and autism … neuroimmune crosstalk has a considerably large impact on brain development during early ontogenesis. … The results propose the relevance of altered synaptic vesicle recycling, cytoskeletal structure and signal transduction” [Gyorffy, et al. 2016]. To put it simply: altered cytoskeletal structure will influence normal developments of both neural and immune systems simultaneously. Research has already shown that cytoskeletal defects are closely related to immunodeficiency diseases [Moulding, et al. 2013] and that the pathogenesis of asthma may be related to the ratio and dysfunction of natural killer T cells [Yan-Ming, et al. 2012]. This point was further confirmed by children born after ooplasmic transplantation [Cohen, et al. 1997]: Seven out of 13 children suffered from allergies and with more of them having other immune problems [Chen, et al. 2016]. Thus, immune problems are another consequence of MRT syndrome.

Cytoskeleton and muscle differentiation

In muscles, cytoskeleton actin filaments slide past myosin filaments toward the middle of the muscle unit sarcomere to produce muscle contraction [Cooper, 2000]. “The heart is the most heavily worked muscle in the body, contracting about 3 billion (3X109) times during the course of a human lifetime. Heart cells express several specific isoforms of cardiac muscle myosin and actin. Even subtle changes in these cardiac-specific contractile proteins – changes that would not cause any noticeable consequences in other tissues – can cause serious heart disease …Familial hypertrophic cardiomyopathy is a common cause of sudden death … another type of heart condition, called dilated cardiomyopathy, which can also result in early heart failure” [Alberts, et al. 2015 P.923]. Thus disturbance of cytoskeleton by SCNT and MRT will likely produce severe muscle and heart problems.

In brief, cells are the basic unit of the organ systems in the human body. The chaos of cellular organelles produced by SCNT and MRT will produce inferior cellular structures (as detailed by points A to G, above), and inferior differentiation of all kinds of cells. These inferior cells produce inferior organs and systems, ultimately producing inferior and subhuman babies rather than normal human babies.

Pathophysiology of MRT stillbirth

The pathophysiology of stillbirth related to MRT can be explained as follows [Prather, et al. 1989; Liu, et al. 2003]: Birth is the most stressful condition for the fetus as it transitions into a baby. Besides high pressure on the fetal head and body for a prolonged time, there are major cardiovascular changes that are triggered by lung respiration. This includes a large increase in pulmonary blood flow in order to replace umbilical venous return as the source of preload for the left heart [Hooper, et al. 2015]. The endocrine and nervous systems are activated to mobilize the respiratory, circulatory, and muscular systems to work much harder in unison to ensure further survival. In MRT related stillbirths, the inferior organ systems produced from the inferior cells failed to work. During stress, the blood vessels composed of these inferior cells would contract abnormally and stop the blood from flowing back to the heart. The inferior heart could not respond normally and heart failure would result, leading to respiratory failure and immediately stillbirth [Zhang, et al. 2003].  Even if live births happen, the MRT babies’ futures will be full of risk - from sudden death (due to lack of normal stress reaction), a multitude of severe diseases (as mentioned above), and adversity for future generations [McGrath and Solter, 1983]. The underlying cause is that their ooplasmic structures were disturbed by MRT, resulting in abnormal cytoskeletal structure in the recipient oocyte and in later differentiated cells of impaired functioning that can be inherited by the offspring. While karyotypes of stillbirth fetuses derived from MRT were normal [Zhang, et al. 2016], genetics only plays one role in the many facets of heredity.

Gene and cytoplasm inheritance

Gregor Mendel first reported discrete “units of inheritance” of phenotypic traits based on experiments differentiating characters of pea plants by fertilization [Mendel, 1865]. The term “gene” was used 16 years later [Johannsen, 1905]. Published models of double-stranded DNA molecules [Watson and Crick, 1953] strengthened the theory of heredity to be related to genes and DNA. Genetics is the study of genes, genetic variation, and heredity in living organisms [Griffiths, et al. 2000]. Some define genetics as the study of heredity, but if genes and DNA were the only materials required for hereditary, genome transfer directly into culture media rather than into ooplasm should produce offspring of the said species. Recognition of hereditary function of cytoplasm is critical.

In the cytoplasm, most organelles are inherited. Mitochondria are essential maternally inherited [Dalton and Carroll, 2013]. The dynamics of a living cytoskeleton are also inherited [Bursac, et al. 2007] by means of self-organization or self-assembly [Huber, et al. 2013]. The endoplasmic reticulum (ER) spreads throughout the eukaryotic cell and is contiguous with the nuclear envelope. ER distribution and dynamics in yeast might be conserved in animal cells [Du, et al. 2004]. Centrosome and centriole are also inherited [Schatten and Schatten, 1986; Wilson, 2008]. Thus, the cytoplasm and its organelles are similar to genes and DNA as they are hereditary materials.

Different inheritance patterns in genes and in cytoplasm

The inheritance pattern of genes and DNA is by replication [koltsov, 1927] accompanied by cell division. During fertilization, both the sperm and the egg contribute equal haploid genomes [Schatten, 1994].

The inheritance pattern of cytoplasm and its organelles in humans is different and is by duplication accompanied by cell division. This is a process in which oocyte centrioles specially reduce in oogenesis (with its components retaining asymmetrically in ooplasm) and restore the zygotic centrosome at fertilization with sperm centriole. The egg contributes the vast majority of the zygote’s cytoplasm [Schatten, 1994].

Cell division (mitosis and meiosis) is a crucial mechanism in heredity and life. If there was no cell division, there would be no cell heredity. If DNA and genes are replicated and the cell is not divided, the DNA and genes cannot be inherited to progeny cell(s). In humans, during each somatic cell cycle, the chromosomes, cytoplasm, and centrosomes duplicate in interphase, and all of them split in two during mitosis [Schatten, 1994]. In oocyte meiosis, human meiotic spindles have centrosomes but no centrioles [Sathananthan, et al. 1991]. Human sperm have centrioles. A well-defined proximal centriole is present next to the basal plate of the sperm head, while the distal centriole gives rise to the central strand of the sperm tail flagellum [Sathananthan, et al. 1991]. Oocyte centriole reduction plays an important role in preventing parthenogenesis and ensures biparental fertilization [Manandhar, et al. 2005]. The maternal γ-tubulin (the component of the reduced oocyte centriole) is often localized at the egg cortex [Gard, 1994], ensuring that the sperm centriole will easily bind to this microtubule-nucleating protein. The zygotic centrosome is the blending of maternal and paternal constituents, with the maternal centrosomal components (γ-tubulin and 25S “γ-some”, etc.) attracted to the paternal “seed” – the proximal centriole of the sperm [Schatten, 1994; Sathananthan, et al. 1991].

Centrosome and heredity control system

The centrosome in cytoplasm was discovered in 1876 [Wunderlich, 2002]. The centrosomes are the command centers for cellular control, in both cell division (cytokinesis) and cell-cycle progression [Doxsey, 2001]. Recently, identification of many kinds of RNA and more than three hundred kinds of proteins in and around centrosomes have highlighted the evolutionary conservation of centrosome functions [Bettencourt-Dias and Glover, 2007; Gadde and Heald. 2004]. The centrosome not only serves as the microtubule organizing center, but also as the actin organizing center, the intermediate filament organizing center, and the Golgi organizing center [Farina, et al. 2016; Goldman, et al. 1980; Alieva, et al. 1992; Schatten, 1994]. Any change in the centrosomes can block or impede cell division [Rappaport, 1986] because they function to organize the astral microtubules to further orient spindle [Khodjakov and Reider, 2001].

Heredity in most species is very stable. Human beings have a history of about five million years of repetitive cell division, and still have conserved a karyotype of 46 chromosomes and an intact cytoplasm. Aneuploidy cells are produced continuously throughout life from our embryonic stage until our death. Yet why are most people not characterized as being aneuploidy or affected by mosaicism? This is because the cytoplasm of eukaryote cells contains several families of protein kinases such as cyclin-dependent kinases (Cdks), polo-like kinases (Plks), Aurora family of kinases [Xie, et al., 2005], and many other families of proteins to form heredity control system. This system include checkpoints and licensing factors for DNA replication [Blow and Dutta. 2005; Shen and Prasanth, 2012; Heichman and Roberts, 1998], for centrosome duplication [Loncarek and Khodjakov, 2009; Lu, et al. 2009; Bettencourt-Dias and Glover, 2007] and for spindle assembly [Musacchio and Salmon, 2007; De Antoni, et al., 2005; Chen, R. H. 2002; Faesen, et al., 2017]. The cycles of centrosome duplication and DNA replication coordinate with each other, and rely on these proteins in the cytoplasm and centrosome for regulation. For example, one of the centrosomal substrates, cdk2, couples centriole duplication to the onset of DNA replication at the G1/S phase transition. Similarly, the G1/S phase regulating proteins include cyclins D and E, cdk4 and 6, cdk inhibitors p53, ZYG-1, Aurora kinases, … etc. These regulate the G1 phase to be finished perfectly before progression into the S phase. Otherwise, the cells would arrest in the G1 phase [Kramer, et al. 2002; Haase et al. 2001]. Additionally, Plks are important mediators for various cell cycle checkpoints that monitor centrosome duplication, DNA replication, segregation of chromosomes, and mitotic exit [Xie, et al., 2005]. In abnormal cells such as aneuploidy cells, there is no mitotic exit to facilitate division. Thus apoptosis will occur in these cells, while normal cells with mitotic exit will continuously divide and grow. Aneuploidy cells can be physiologically normal in embryonic stages for special functions of differentiation and implantation. Most of the aneuploidy cells in human embryos do not contain heredity characteristic due to perfect heredity control system in their cytoplasm to stop mitotic exit. Hereditary diseases are the concurrent defects of both DNA and hereditary control system. When the coordination between the cell cycle and the heredity control system is damaged, pathological aneuploidies or cancer will occur (such as hydatidiform moles and choriocarcinoma) [Bettencourt-Dias and Glover, 2007; Prasanth, et al., 2004]. The heredity control system is very meticulous. Any cell being divided should pass the checkpoints and licensing system (listed above) from at least 10 to upwards of 30 times. In human embryos of in vitro fertilization (IVF), the heredity control system functions much more effectively than the Preimplantation Genetic Screening (PGS) that is performed in some IVF clinics [Cui, 2017d]. The natural heredity control system allows lesser than 1% of live births to contain genetic abnormalities, with true mosaicism comprising less than 0.5% [Capalbo, et al., 2017]. However, in PGS, the patient’s history (which is closely related how perfect of the patient’s heredity control system to be) is neglected. Thus, PGS is lack of any predictive effect in clinical diagnosis. During PGS, the embryo cytoskeletons are damaged by repeated temperature changes (such as freezing) [De Storme, et al., 2012; Gomes, et al., 2012; Semmrich, et al., 2007], by toxic cryoprotectant (Dimethyl Sulfoxide), and by invasive mechanical or laser techniques that can lead to artifact results [Sachdev, et al., 2017]. The damaged cytoskeleton not only produces a new aneuploidy, but also interferes with normal neuron growth (mentioned above) and mental development (as seen in mice and human experiments) [Kahn and Baas, 2016; Yu, et al. 2009; Middelburg, et al. 2011]. When SCNT and MRT are performed, the heredity control systems of the normal cell heredity are thoroughly torn and destroyed by spindle extraction.