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From Embryology

Week11

link to permalink image Duct cochlear duct The cochlear duct is an fluid filled cavity inside the cochlea. It located between the tympanic duct and the vestibular duct, and between the basilr membrane and reissner's memebrane. It derived from otic placode, otic vesicle, and originated from surface ectoderm.

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Sensory - Hearing and Balance Development --Inner Ear

Technical Progression

Three-person in-vitro fertilization is a process where a small proportion of genetic information encoded within mitochondria are replaced to prevent mitochondrial disease passing through generations. Main approaches to achieve this goal involve the replacement of mitochondrial genome between gametes or embryos [1] [2].

  • The first proposed treatment is cytoplasmic transfer, which transfers a small part of ooplasm from one oocyte to another. however, this approach are then considered to be inadequate to prevent the inheritance of diseased mitochondrial. because it adds in donor mitochondria without removing the mutated mtDNA, which will then generate a 'heteroplasmic oocyte' with both mitochondria haplotypes [3].
  • New emerged approaches of mitochondrial transmission are pronuclear transfer(PNT), spindle transfer (ST) and Polar body transfer (PBT). however, none of this techniques have been proved on generating healthy human offspring due to the technical difficulty, as well as the ethics issues being recognized worldwide [4].
  • Major breakthroughs of these techniques rely on the practice on animal models (mice and primate) [5], early stage human embryo and stem cell studies [6].


Image Source: http://www.popsci.com.au/science/medicine/what-3parent-babies-mean-for-the-future-of-reproductive-medicine,400376

Cytoplasmic transfer

Cytoplasmic transfer is also named Ooplasmic transfer. It is an in vitro fertilization (IVF) technique,which introduce a small amount of ooplasm from a donor oocyte or zygote into compromised oocyte or zygote from patients.

Why do cytoplasmic transfer?

The quality of the oocyte cytoplasm is critical for the future of the embryo [7].Following ovulation, the survival of zygote depends almost exclusively on maternal messenger RNA and proteins that accumulated during oocyte growth and maturation within the ooplasm. it is until the maternal-to-zygotic transition (MZT) stage during the 4–8‐cell stage in humans where the new zygote genome is activated and replace the maternal cytoplasm to be predominant in regulating the zygote development [8] . the maternal transcripts are thus responsible for the first few cleavage divisions and for transition of the maternally controlled zygote into an activated embryonic genome [9].

Researches are still underway to investigate the molecular and cellular mechanisms how ooplasm regulates the maturation and activation of human oocytes and zygotes[10]. The ooplasmic factors involved in this regulation are messenger RNA, maternally stored proteins, stockpiles of energy substrates, other energy-production components and many factors yet to be determined. The benefits of ooplasm transfer are revealed by the following two hypothesized biochemical mechanisms: correction of a putative imbalance between anti-and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential[11].

What is the procedure?

Cytoplasmic transfer can be performed either as a repair of oocyte or repair of Embryo [12]

In method one, the cytoplasm is withdrew from a donor’s oocyte, and then injected into a patient’s oocyte together with the sperm cells which will then fertilize the oocyte. In Method two, the cytoplasm from donor is injected into a patient’s fertilized oocyte.

Diagram of cytoplasm transfer [13]
Simplified Cell Structure
Egg repair by Cytoplasmic transfer
Embryo repair by Cytoplasmic transfer

Key events of Cytoplasmic Transfer

  • 1982, United Kingdom - Audrey Muggleton-Harris's group at MRC Laboratory Animals Center in Surrey, developed the technique and reported the first successful mammalian cytoplasmic transfer in mice [14].
  • 1982 United Kingdon - Muggleton-Harris's group transferred cytoplasm from mice strains whose oocytes divide past the two-cell stage in vitro into mice to overcome the two-cell barrier [15].
  • 1997, United States - Jacques Cohen, Richard Scott, Tim Schimmel, Jacob Levron, and Steen Willadsen at the Institute for Reproductive Medicine and Science of St. Barnabas in West Orange, New Jersey, announced the birth of a baby girl after the first successful human cytoplasmic transfer [16].
  • 1998 United States – The US Food and Drug Administration (FDA) banned the procedure.
  • 2002 United States - one of the children conceived through ooplasmic transfer were diagnosed with pervasive developmental disorder, and indicated mild developmental delays to severe autism.
  • 2014 United States - public meetings to discuss mitochondrial manipulation techniques were held by FDA. There was no formal decision made base on the efficacy of Cytoplasmic transfer, but agreements were made on further practice on animal models to provide scientific data.
Cytoplasmic transfer cases in human[17]
Type of Cytoplasm transferred to recipient oocytes No. of Procedures Pregnancies achieved Offspring delivered
Synchronized fresh oocytes by electrofusion [18] 3 0 0
Synchronized fresh oocytes by injection (USA) [19] [20] [21] [22] [23] 30 13 16
Synchronized fresh oocytes by injection (Israel) [17] 15 5 6
Synchronized frozen oocytes by injection [24] 4 1 2
Asynchronous 3-PN zygotes by injection [25] 9 4 5

Risk of Cytoplasmic transfer -- Heteroplasmy

Heteroplasmy is defined as the mixture of more than one Mitochondrial DNA (mtDNA) type within the cytoplasm of an individual [26] [27]. It is believed to have rare heteroplasmic mutation in healthy individuals previously. however, human mtDNA sequencing has now showed that each person has some low- frequency, slightly different mtDNA types mixed with the maternally inherited dominant type. and this low-frequency variants arise from mutations during growth and mitosis of individual cell. The two types of heteroplasmy are length heteroplasmy and sequence (or site) heteroplasmy [28] .

An example of sequence heteroplasmy visualized by partial mtDNA sequencing [29]
  • Length heteroplasmy is the presence of mtDNA molecules that differ in length.
  • Sequence (site) heteroplasmy is the presence of mtDNA molecules that have different nucleotides at the same site.


Although the low frequency mtDNA mutation is quite common and cells can contain varying proportions of mutated and wild-type mtDNA [30]. Cells can usually tolerate the level of mutations. Only if the mutation is pathogenic, and the percentage of variants exceeds the biochemical threshold, defects will be induced[31].

Cytoplasmic transfer in IVF procedure has the risk of manifesting the mutations as it combines the mtDNA from donor with the maternally inherited mtDNA of the recipient. Heteroplasmy is thus one of the major concerns arise regarding cytoplasmic transfer in IVF procedure [32]. Severe disease can occur due to heteroplasmy in the offspring’s mitochondria. They may affect the development of the muscle, brain and endocrine system [33]. They could also result in mitochondrial disease developing either in the child or in future generations [34].

Spindle-chromosome transfer

Spindle-choromosome transfer is a modified cloning technique which transfers the meiotic spindle and attached chromosomes (spindle-chromosome complex, SCC) from one mature oocyte to another to select for a cytoplasm or mtDNA background [35]. Comparing to cytoplasmic transfer, spindle transfer can reduce the risk of heteroplasmy, thus offer a better reproductive option to prevent mtDNA disease transmission in affected families [36]. has better potential in This technology has been used to generate both cattle and mice after subsequent fertilization (Bai et al, 2006, Bao et al, 2003, Wakayama et al, 2004 and Wang et al, 2001), and has generated live monkeys (Macaca mulatta) after sperm injection [37]. Spindle transfer between human oocytes has also result in blastocyst development and embryonic stem cell derivation with very low levels of heteroplasmy [38].

PMID 25229667 PMID 23103867 PMID 24382342

What is the procedure?

  1. Assisted reproductive technologies are used to extract the intending mother’s egg from her ovaries. The cytoplasm of the intending mother’s eggs contains the unhealthy mitochondria.
  2. Chromosomes, the nuclear DNA material, are found in the intending mother’s eggs are grouped together in a spindle-like formation. The chromosomes are removed for transfer to the donor egg. The chromosome-free egg, which contains the unhealthy mitochondria, is then discarded.
  3. Separately, a donated egg is also extracted from an unrelated woman who has healthy mitochondria. Similarly, the chromosomes of the donor’s egg are removed. However, these chromosomes are discarded, leaving behind the healthy mitochondria in the cytoplasm.
  4. The spindle-like chromosomes previously taken from the intended mother’s egg are inserted into the enucleated donor’s egg.
  5. The resulting reconstructed egg contains nuclear DNA from the mother and the healthy mitochondria from the donor.
  6. The resulting egg can now be fertilized with sperm from the intended father. The resulting embryo will be implanted into the intending mother and will develop unaffected by inherited mitochondrial disease.
Diagram of spindle-chromosome transfer [39]
[[File:|200px|thumb|Left|Simplified Cell Structure]] [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]] [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]

(reproduced diagrams to be uploaded)

Primate model

Due to the uncertainty of the health risks related to spindle-chromosome tranfer, experiments in non-human primates are required to access the safety of this procedue. Tachibana et al(2009) have carried out maternal spindle transfer using healthy eggs from non-human primates (rhesus macaques)[40].
  • a - removed the nuclear material plus a cellular membrane (a karyoplast) from a mature oocyte, leaving behind its mitochondria. The nuclear material in the karyoplast consists of condensed chromosomes attached to thread-like spindle fibres (the spindle–chromosomal complex).
  • b - transferred the karyoplast to an oocyte whose nucleus had been removed (a cytoplast).
  • c - fused the karyoplast with the cytoplast and then fertilized the reconstructed oocyte.
  • d - developing blastocyst was implanted in a surrogate mother.
  • e - mother gave birth to a healthy baby.

Some of the resulting embryos were successful and produced healthy offspring with low mtDNA carryover. However it is still too early to determine whether spindle-chromosome tranfer is a safe procedure. Because defects may develop later in life, or in their own offspring. Thus long-term studies are required to access the effects of this procedure, which includes life-long monitoring and multi-generational tracking.

Primate model of spindle-chromosome transfer [41]

Current Research

Currently, researchers at Newcastle University in the United Kingdom are collaborating with the Oregon researchers who successfully generated the first primate model in 2009. They are testing the maternal spindle transfer technique on human oocytes. Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA. Thus they concluded that the mtDNA can be efficiently replaced in human oocytes, although some ST oocytes displayed abnormal fertilization[42].

Pronuclear transfer

Pronuclear Transfer is similar to Maternal Spindle Transfer but performed as a repair of embryo. it fertilizes the mother’s egg first and then transfers the nuclear DNA to the fertilised donor egg containing healthy mitochondria, from which the original nuclear DNA has been removed [43].

  • pronuclear transfer procedures was first performed on mice in the 1990s, suggesting the possibility of preventing the transmission of mutated mitochondrial DNA [44].
  • In 2003 scientists at Sun Yat-Sen University in China first attempted this procedure on human embryos. Five genetically modified embryos were implanted into a 30-year-old woman. She became pregnant with triplets, and doctor removed one to give the other two foetuses better chance of survival. After some months, the woman suffered miscarriages and lost both foetuses [44].
  • In 2010 researchers at Newcastle University reported that pronuclear-transferred human embryos developed normally to the blastocyst stage in six to eight days, this marked the procedure as a success in preventing mitochondrial disease [45].

What is the procedure

The nuclear genome from the pronuclear stage zygote of an affected woman is transferred to an enucleated donor zygote [44]

  1. It begins with creating an embryo using the parents’ sperm and eggs.
  2. At the same time, a second embryo is created using a donor egg with healthy mitochondria and the father’s (or donor) sperm.
  3. The pronuclei are removed from the single-cell stage embryo (day one). The leftover enucleated embryo with diseased mitochondria is discarded.
  4. The pronuclei of the second embryo are removed and discarded.
  5. The parents’ pronuclei can be placed into the second embryo for development.
  6. The developed embryo will then be transferred into the mother.


Diagram of pronuclear transfer [46]
[[File:|200px|thumb|Left|Simplified Cell Structure]] [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]] [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]


(reproduced diagrams to be uploaded)


PMID 25763399

Human Embryo Model

Research Group in New Castle University performed pronuclear transfer on human mebryo model[47]:

  • Pronuclear transfer was performed using abnormally fertilised human zygotes generated following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI).
  • Abnormal zygotes were identified on day 1 of development by the presence of one pronucleus (unipronucleate) or three pronuclei (tripronucleate) 18-19 hours after insemination.
  • Karyoplasts containing pronuclei and surrounding cytoplasm were removed from the donor zygote using a biopsy pipette and transferred to a recipient zygote.
  • Following fusion, the reconstituted zygotes were either cultured for 6-8 days to monitor development to the blastocyst stage or were cultured before being disaggregated for analysis of mtDNA in individual blastomeres'.

The safety effects of this procedure (zygote mtDNA carry-over) were tested by sequencing of non-coding control region. The sequence of donor and recipient mtDNA were then compared. Based on the data provided, they believe pronuclear transfer has the potential to prevent the transmission of mtDNA disease in humans. However, Further studies are required to ensure the safety of different techniques when modifying human oocytes and zygotes due to the potential of causing chromosomal or epigenetic abnormalities


Current research on pronuclear transfer Healing Broken Batteries – A short film about mitochondrial disease and the new techniques being developed at Newcastle University.


Polar body transfer

Polar bodies are small cells formed during the meiotic reductive division of the oocyte. they contains complementary choromosomes (to the mature oocyte) and small amount of cytoplasmic segregation[48].

  • Polar body 1 is formed and released during ovulation. it contains a diploid set of chromosomes.
  • Polar body 2 is formed during fertilization and can be identified in the zygote. it contains a haploit set of chromosomes.
  • Both polar bodies are unable to be fertilized and disintegrate eventually

It has been reported with mice modle that coupling Polar body transfer with Pronuclei transfer or Spindle-choromosome transfer may increase the yield of reconstructed embryos with low mtDNA carryover. [49]

What is the procedure

PMID 25472922 PMID 16317618


Diagram of Polar body transfer [50]
[[File:|200px|thumb|Left|Simplified Cell Structure]] [[File:|500px|thumb|middle| Egg repair by Cytoplasmic transfer]] [[File:|500px|thumb|right| Embryo repair by Cytoplasmic transfer]]


(reproduced diagrams to be uploaded)

Mice Model

PMID 24949971 Polar body genome transfer for preventing the transmission of inherited mitochondrial diseases [51] The authur adopts polar body transfer to prevent the transmission of mtDNA variants. they also compare the effects of different types of germline genome transfer, including spindle-chromosome transfer, pronuclear transfer, and first and second polar body transfer, in mice. Their pre-clinical model indicate that polar body transfer has better potential in preventing the inheritance of mitochondrial diseases.

Other approaches

germinal vesicle nuclear transfer

ETM transition

PMID 20943648 PMID 21559368 PMID 21447558

Talk

  • Timeline of The artificial reproductive technology revolution
  • 1984 donor egg pregnancies
  • 1984 frozen embryos
  • 1960 Ovulation induction
  • 1960 Donor sperm
  • 1978 IVF
  • 1988 Pre-implantation genetic diagnosis
  • 1992 Sperm injection
  • 1994 Blastocyst development
  • 1998 Reliable sperm sex selection
  • 2000 Stem cells and cloning and gene transfer
  • 2005 Ovarian reserve testing
  • 2008 Oocyte freezing, social freezing
  • 2013 Whole genome screening of embryos
  • Process of IVF
  • Ovarian hyperstimulation
    • two phase during menstrual cycle: lacteal phase and follicular phase
    • About 30 Follicles grows to large size in every mentrual cycle, but only one survived to the ovulation.
    • FSH drove the growth of follicles, also control the growth of follicles (pituitary down regulation), used to stimulate the mature of multiple follicles (10-30).
    • Estrogen....
    • Monitoring by transvirgena ultrasound.
    • LH containing hormones injected to women to stimulate ovulation.
  • Ooocyte Pick-Up(OPU)
  • Sperm retrieval via ejaculation or surgery
  • In Vitro Fertilisation
    • intra-cytoplasmic sperm injection (ICSI)
    • Embryo culture
    • day1- form Zygote
    • day3- divide to 8 blastomeres
    • day5- frozen, and transfer to uterus
  • Transfer of the embryo(s)
  • New technologies
    • Preimplantation genetic diagnosis(PGD) of embryo. eg, fibrosis-premature death
      • Embryo Biopsy for PGD
      • analysis of blastomeres for PGD
        • FISH
        • PCR
      • What to be detected in an embryo
        • Aneuploidy
        • trisomy 21
        • translocations
        • single gene defects(eg cystic fibrosis)
    • Ovarian reserve and the natural cycle
      • blood test measure hormones(AMH, AFC)
      • Egg banking ('social freezing')
    • Fertility preservation for cancer
    • Oocyte in vitro maturation (IVM)
      • putting oocytes in vitro and culture 24 hours before IVF (Complex medium , protein, FSH, EGF, LH, E2 cysteamine)
      • comparing to conventional IVF, no Hormones required to give to mother for the maturation of oocyte.


Reference

Template:Reflist

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