APPLICATIONS

Nuclear Transfer In Research

Applications

Nuclear Transfer In Research

IVM and nuclear transfer in animal GV oocytes

When immature oocytes are retrieved at the GV phase, they are cultured in vitro and allowed to mature to the fertilizable phase of MII [60, 78-80]. This process is referred to as in vitro maturation (IVM). Nuclear transfer with immature oocytes in animal models is a good system to study the nuclear-cytoplasmic relationship underlying meiosis [84], as well as the process of in vitro maturation, cell cycle and fertilization [56]. Interestingly, GV studies in the mouse system have evidenced that the cytoplasm rather than nucleus is the determining factor for the structure and size of the meiotic spindle [85]. Two other studies used xeno-transfer between mouse and human oocytes to confirm the notion that the cytoplasm determines the structure and size of the meiotic spindle [19, 43].

Basic research studies with mouse GV and MI oocytes have evidenced for oocyte size regulation during IVM via glycine accumulation with the GLYT1 transporter [86]. Mouse GV do not transport glycine until they are removed from the follicular environment and enter the MI phase, at which point the oocyte is concomitantly released from adhesion to the zona pellucida and starts decreasing in size significantly (up to 20%) while causing formation of the perivitelline space (PVS). Unwanted reduction in size is regulated by activating the GLYT1 transporter to uptake glycine and control cell volume. Early formation of PVS has been linked to a significantly higher rates of meiosis completion (80% versus 5.5% if no PVS formed) [87]. Mouse GV continuously uptake glycine until MII, when endogenous glycine is detected at high intracellular levels [86, 88].

Furthermore, mouse studies have evidenced for the importance of cumulus cell contact to enable the positive effect of gonadotropins on oocyte maturation during in vitro culture [44]. Contact with cumulus cells during in vitro maturation results in dramatically higher rates of blastocyst formation. In vitro matured GV that are enclosed in a cumulus-oocyte-complex undergo maturation, fertilization and blastocyst formation at similar rates to in vivo matured oocytes. This is in contrast to denuded GVs which complete the developmental stages at much poorer rates, and those that do reach the blastocyst stage exhibit aberrant expression of the pluripotency factor Oct4 [44].

Previous mouse studies have evidenced for a high success rate of in vitro maturation of mouse oocytes that have undergone GV transfer [18, 40, 89]. Some mouse studies have suggested that the cytoplasm of young mice cannot rescue age-associated meiosis defects of GV from aged mice [90]. Other mouse studies of GV transfer of old oocytes into the cytoplasm of young oocytes have shown up to ~20% rescue of meiotic defects in the old nuclear material chromosome misalignment [91]. Research from the Darwin Life team has shown that meiotic defects of in vitro aged oocytes can be rescued via GV transfer [38]. Further research from the Darwin Life team shows that MII nuclei generated from GV transfer are functionally competent to support preimplantation development [41], and that they can produce live birth offspring [37].

Only the cytoplasm at the GV phase rather than at the MII or zygote stages can support GV meiotic maturation [18]. Most importantly, only in vivo mature cytoplasm can successfully support the development of the in vitro matured MII nucleus [17, 37, 39, 42, 44]. In vitro matured oocytes have reached nuclear maturation but not cytoplasmic maturation [60], and are developmentally incompetent and unprepared for fertilization and embryonic development, due to lack of cytoplasmic factors required to develop the MII spindle into a fully functional pronucleus [17]. These studies in the mouse system evidence for the necessity of a second nuclear transfer step so that preimplantation development can be completed [17, 37, 39, 41]. This step is also crucial for the significant dilution of carryover maternal pathogenic mtDNA.

IVM and nuclear transfer in human GV oocytes.

Historically, IVM was considered for women who are sensitive to gonadotropins or have diminished ovarian reserve [25]. Women suffering from polycystic ovary syndrome (PCOS), representing 5-10% of women of reproductive age [78], have many antral follicles (2-8mm diameter) and traditionally benefit from IVM in the course of their IVF protocol [79]. Women who either respond poorly to ovarian stimulation or are at high risk of ovarian hyperstimulation syndrome (OHSS), such as women with PCOS, benefit from having their immature oocytes undergo IVM as an initial step in their IVF protocol [79]. Finally, IVM has been proposed for fertility preservation for patients with cancer who are unable to undergo hormonal stimulation and will experience loss of ovarian function after chemotherapy and radiation [79], and for a treatment for women with infertility [60, 78]. The first human birth resulting from the in vitro maturation of a GV was reported in 1991 [92]. In 2004, pregnancy rates with IVM were 30-35% per retrieval with 10-15% implantation rates [60]. Currently, over 4000 children have been born from IVM without any health problems [79].
In traditional IVF cycles, immature oocytes at the GV phase are not fertilizable and do not qualify to proceed with the normal protocol. Immature oocytes and their cumulus cells are removed from follicles (typically 2-13mm diameter [93]) before ovulation occurs, and are cultured for up to two days in vitro to mature to a fertilizable state [78-83]. During this culture, nuclear maturation begins spontaneously, due to the removal of an inhibitory factor present in the follicular context [81]. For human oocyte maturation, studies using different in vitro maturation culture conditions report a wide range between ~40% to ~80% maturation rate for GV oocytes [45, 78-81, 83, 94-96], and rarely 100% maturation rate [97, 98]. Other than the contents of the maturation media, major factors affecting oocyte maturation rate are: maternal age (see Background above), stimulated versus non-stimulated ovaries, the size of the oocyte at the time of collection, and the presence of the cumulus-oocyte-complex (COC) [44, 60].
GVs from stimulated cycles reach the MII stage in 20-24 h and maturation outcomes are superior to non-stimulated cycles, where GVs reach the MII stage 36-38 h after IVM [60]. Basic research studies have suggested that the size of the oocyte at the time of collection impacts the rate of maturation [99], and that normally fertilizable oocytes after IVM are significantly larger compared to in vivo mature oocytes from standard IVF cycles or PCOS-ICSI cycles [100]. Day 0 GV oocytes are smaller (106-111 µm) compared to in vivo matured Day 0 MII oocytes (110-120 µm), but they have a tendency to grow during the in vitro maturation process (additional 3µm on average for GVs from traditional IVF cycles, but no growth for GVs from unstimulated PCOS patients) [99]. Furthermore, Day 0 GVs are suggested to require a minimum 100 µm diameter to reach MII [99]. Mouse studies have suggested that oocytes regulate their volume via glycine uptake and osmotic support through the GLYC1 transporter [86, 88]. Although this has not been confirmed for human oocytes, research with human embryos has shown that a high level of glycine turnover correlates with high rates of implantation and pregnancy [101].

Importantly, basic research studies with human oocytes have confirmed the results with animal models that the in vitro matured oocyte is not developmentally competent. Maturation to the MII stage that is competent to support preimplantation development involves structural changes in the cytoplasm including an increase in the number of mitochondria, modifications to the Golgi apparatus and accumulation of ribosomes [19]. The prolonged culture during human IVM leads to a significantly reduced protein content in the matured oocyte compared to in vivo matured oocytes [83], and there are more than 2000 abnormally expressed genes in IVM oocytes compared to the in vivo matured oocytes [102]. Furthermore, prolonged IVM culture causes the mitochondria-smooth endoplasmic reticulum aggregates to be largely replaced by small mitochondria-vesicle complexes [103].
Although the human oocyte does not have luteinizing hormone (LH) receptors, MI division is triggered by the LH surge in the menstrual cycle (or gonadotropin injection in IVF cycle) [56]. The LH surge initiates meiosis indirectly by interacting with the cumulus cells which have LH receptors and are connected to the oocyte ooplasm via gap junctions. Cumulus cells are important for oocyte maturation not only due to enabling LH-mediating meiosis initiation, but they also influence the nutritional environment by converting glucose or lactate into pyruvate to be used by the maturing oocytes [60]. Studies in mouse oocytes undergoing in vitro maturation have evidenced for the importance of the site of maturation (in vitro versus in vivo) [17, 37, 39, 42, 44] and for the important role of cumulus cells enclosure during in vitro maturation [44].
Whether the size of the GV follicle affects the subsequent rate of in vitro maturation is still unclear. Studies on follicle size suggest that the minimum follicle size required for developmental competence is 5mm [104]. However no significant differences are reported in the diameter of MII oocyte siblings, their rate of fertilization and generation of good quality embryos, when their lead follicle is either <10mm or >10mm; the same is true for immature oocyte siblings of dominant follicles that are ≤10mm, 10-14mm, or ≥14mm, in which cases sibling GV oocytes from all groups exhibit similar maturation rates, fertilization rates, and good quality embryos [105, 106].
Nuclear transfer technique has been applied to human gametes at the GV [19, 25, 45], MII [13, 27, 28, 33, 34] and zygote (2PN) [29, 32, 46] stages. A milestone study conducted by the Darwin Life team showed that transferring of GVs from old patients (>38 years old) to the ooplasm of young patients (<31 years old) results in 80% normal MII spindles, suggesting that GV transfer may be developed into a procedure to treat age-related aneuploidy [45]. GVT involves several key steps, with an overall success rate reported as 20%, and a subsequent IVM rate of ~60% [45]. Successful human egg reconstitution for the elimination of maternal mtDNA carryover will require subsequent transfer of the in vitro developed MII spindle to an enucleated in vivo MII oocyte.

Applications in clinical investigations and FDA regulations

Egg reconstitution with two sequential nuclear transfers can be potentially applied to women with high levels of pathogenic mtDNA who wish to reduce the risk of NT carryover to minimal levels. In traditional IVF treatment, clinicians retrieve mostly developmentally mature (MII) oocytes for IVF procedures, but in the case of preventing pathogenic mtDNA carryover, oocytes can be retrieved at an earlier developmental stage (GV), before meiotic maturation, and put through a protocol of two sequential transfers.

Related to total cytoplasmic replacement, a micromanipulation technique called ooplasmic injection has been successfully applied for women with a history of poor quality embryos [56, 107]. In this technique, up to 15% of cell volume is injected to the oocytes of patients with a history of fragmentation and poor embryo quality. Such ooplasmic provision of functional, healthy mitochondria and key cell constituents has led to several successful, healthy pregnancies.

Using a specific nuclear transfer technique, Darwin Life aims to provide research evidence for total cytoplasm replacement. Darwin Life operates under an IRB-approved research license and our goal is to develop reconstitution protocols for age-related infertility. The nuclear transfer technique involves sequential GV transfer and Spindle transfer, resulting in the egg carrying the mother’s own nuclear DNA but with a fully replaced, healthy cytoplasm.