Perspective Chapter: Application of Abnormally Fertilized Eggs and the Associated Clinical Outcomes

Abstract

Human IVF laboratory often shows many abnormal fertilization eggs, such as no pronucleus (0PN), monopronucleus (1PN), three pronuclei (3PN) or multi-pronuclei (mPN) zygotes and these abnormal eggs are usually not used and typically discarded. Recent researches have showed that further evaluation on these abnormal eggs may provide some hope for aged infertile couples to have babies in their family. Our study showed that 0PN eggs may be rescued by introcytoplasmic sperm injection (ICSI). The 1PN zygotes should be cultured to observe their cleavage and blastocyst formation until Day 3 to Day 6. Selecting normal chromosomal embryo transfer may have healthy baby birth. Although most of 3PN embryos are genetically abnormal chromosomal composition, a small portion of 3PN embryos may develop to blastocyst with normal chromosomal composition. Also, those embryos derived from 3PN, especially by ICSI, have more possibility for self-correction to become normal euploid embryos. The microsurgically removing 1PN from 3PN zygotes may artificially correct this abnormal fertilization. After one PN removal, the formed blastocyst may be screened by the PGT for embryo transfer in rare embryo patients to achieve pregnancy and delivery of a healthy newborn. Based on no obvious difference of ooplasm between normal fertilized 2PN and 3PN zygotes, the cytoplasm of 3PN zygotes may be used to supplement the aged woman poor oocytes to improve embryo quality. Transferring partial cytoplasm from 3PN zygote to the fertilized 2PN zygotes of aged woman may promote the receipt embryo to develop blastocysts. This partial ooplasmic transfer does not change the aging woman genetic composition and the woman embryos still keep her with her husband genetic genes in the cell nucleus. However, the baby born with this technique might appear epigenetics because the mixed mitochondrial DNA would be passed on to all future generations.

1. Introduction

Assisted reproductive technology has been widely used for the treatment of infertile couples to realize their dream of having a baby. Currently, this technology mainly contains in vitro fertilization (IVF) and its related procedures—intracytoplasmic sperm injection (ICSI). The key point of this technique is that the patient needs to be administered with some medicine to stimulate the ovaries to produce more oocytes during one reproductive period cycle and the oocytes on ovaries will be retrieved. After oocyte retrieval, insemination is performed with the use of conventional IVF or intracytoplasmic sperm injection (ICSI) under the laboratory condition. In all IVF laboratories, normal fertilization is determined by the presence of two pronuclei (2PN) associated with extrusion of the second polar body at 16–18 h post-insemination. Those zygotes displaying successful extrusion of the second polar body and two even pronuclei (2PN) are considered to be “normally fertilized,” and they are cultured further to observe embryo quality and development. An absence of either 2PN or the second polar body indicates fertilization failure in which eggs show zero pronucleus (0PN). In the other way, those zygotes showing a single or more than 2PN are considered to be “abnormally fertilized.” Sometimes, three or more PN are visible in the cytoplasm of the oocyte at the fertilization check, which indicates either polyspermy or failure of extrusion of the second polar body. Occasionally, only 1PN is seen in the cytoplasm of the oocyte, and this is generally attributed to oocyte parthenogenetic activation, irregular pronuclear formation resulting from asynchrony of pronuclear appearance, or possibly male and female pronuclear fusion. Zygotes with one PN are thought to be at a higher risk of being haploid, and the transfer of these embryos is expected to result in an implantation failure [1]. Even though the zygotes deriving from abnormally fertilized oocytes are capable of normal in vitro development, they are usually discarded because of a higher risk for abnormal ploidy constitution (i.e., haploidy, triploidy, or tetraploidy) [2]. Overall, 3 ∼ 10% of inseminated oocytes fertilize abnormally, and the embryos deriving from them are typically discarded in the absence of a reliable approach to monitor their genetic risk for ploidy defects [3]. However, recently, some research analysis has showed that abnormally fertilized oocytes have given development potential and can produce healthy live births [4, 5, 6, 7]. In order to evaluate character of abnormal fertilization eggs, this chapter will concentrate on discussing the application of unfertilized and abnormal fertilization oocytes.

2. IVF failure and abnormal fertilization

Currently, human IVF technique is widely used in treatment of infertile couples to realize their family desire to have a child. However, in vitro fertilization is a complex process that requires many steps, which involves the combination for normal sperm with normal oocyte. As we know, all cells in the human body consist of two copies of each chromosome in 23 pairs of chromosome: one set is from the mother, and another set is from the father. The gametes (sperm and oocytes) contain only one copy of the chromosomes. Thus, both oocyte and sperm are haploid. During oocyte maturation, the oocyte needs to go through meiosis and to repel its excess chromosomes by releasing polar bodies to form metaphase II (MII) oocytes. The first polar body is extruded around the time of ovulation or oocyte retrieval. The presence of the first polar body indicates the oocyte mature situation that has the potential to be fertilized. Only matured oocytes can be fertilized. Germinal vesicles (GVs) are immature oocytes that do not have the ability to be fertilized. Atretic oocytes or oocytes with broken zonas are not viable and are unable to be fertilized [8, 9].

Under normal standard situation, the sperm after capacitation must have the ability to reach a mature oocyte (egg) and enter the oocyte, activating matured MII oocyte to form pronucleus. At the same time, the oocyte must have ability to block more than one sperm to enter oocyte to avoid to form polyspermy [10]. If sperm does not have ability to move or penetrate oocyte zona pellucida, the intracytoplasmic sperm injection (ICSI) technique can overcome this obstacle.

If it is normal fertilization, one prenucleus (PN) is from the oocyte, and another is from the sperm. The oocyte and sperm are haploid, in which each contains only 1/2 of the normal genetic material of a human, and their combination recover their adult body chromosome numbers. During this fertilization process, one important condition is that oocyte must be matured MII, and sperm must have ability to enter eggs. Also, sperm must activate oocytes to form pronuclei. When the sperm enters the oocyte, its head must de-condense, allowing the chromosomes to unwind. The membrane surrounding the sperm must be broken for the sperm head to de-condense [11]. This is the normal traditional IVF process when the sperm tail detaches from the head. However, ICSI needs to bend the sperm tail using the ICSI pipet by breaking the membrane surrounding the sperm. Thus, normal fertilized oocytes or zygotes should contain two prenuclie (2PN) with two polar bodies at 16–18 hours post-insemination. Time-lapse monitor has revealed 17 ± 1.0 hour after insemnination as the best observation for fertilization [12]. However, routine human IVF laboratories always have many abnormal fertilization eggs including zero PN (0PN) unfertilization, 1PN, 3PN, and multi-PN phenomena (Figure 1). Abnormal fertilization may be due to a number of reasons. For example, a 1PN could be caused by the failure of the sperm to de-condense or by failure of the oocyte to activate. 3PNs can be caused by the failure of the oocyte to block more than one sperm from entering the oocyte or by the failure of the oocyte to extrude the second polar body [13, 14].

Figure 1.

Human oocytes show several different features after insemination: Unfertilized egg (0PN), normal fertilization (2PN), abnormal fertilization (3PN, multi-PN).

Because these unfertilized oocytes and abnormal fertilized oocytes have no potential for producing a viable pregnancy, they are immediately discarded in most human IVF laboratories. However, recent many studies have found that some abnormal pronuclear-stage embryos are not necessarily genetically defective and have potential development capability to become blastocysts [7, 15]. Furthermore, normal pregnancies derived from monopronulcear (1PN) zygotes have been reported [4, 16, 17]. Abnormal fertilization oocytes may produce healthy babies [4]. Thus, the exploring application of these abnormal eggs will be significant for rare egg patients. We have drawn an outline for the development and application of these abnormal fertilization oocytes (Figure 2).

Figure 2.

After fertilization, the tendency of oocyte development shows no fertilization (0PN), Normal fertilization (2PN), and abnormal fertilization (3PN, multi-PN). 0PN and 1PN oocytes may be further cultured to cleavage stage and blastocyst stage. Normal 2PN embryos may be used for transfer or cryopreservation. 3PN zygotes may be enucleated for cytoplasmic transfer or one pronucleus may be enucleated from 3PN eggs to form normal 2PN embryos. Multi-PN oocytes should be discarded immediately. Abnormal fertilization embryos should be diagnosed by PGT to select normal chromosome composition embryos for transfer.

3. No pronuclear (0PN) oocytes

At fertilization check, the embryologist usually observed that some of the eggs have no fertilization, which shows 0PN. This often presents two situations. One is immature oocyte without a polar body or staying germinal vesicle stage, which showed that these immature oocytes will not be capable of fertilization at a later time. Further, those oocytes usually do not have potential development capability on the next day. However, those MI oocytes without polar body at the oocyte retrieval day may be cultured for an extended time to observe their polar body appearance. After 5–6 hour culture, the oocytes with polar body oocytes may be used for ICSI to get fertilized zygotes.

Another situation is no fertilization with mature oocytes that have not shown the characteristic formation of their pronucleus. This may have either sperm or oocyte defect in its number of chromosomes, or they do not have a complete plan for building an embryo. Some eggs in the ovaries are atretic and nonfunctional, and have no chance of developing into an embryo. Even though these oocytes are inseminated with normal sperm, they cannot show fertilization with 0PN [18], which results in the egg being incapable of further development. The sperm can also be the cause of a mature egg not fertilizing [19]. The semen sample may show a low number of sperm and abnormal morphology, and these sperm are incapable of penetrating the egg’s membranes. This is often associated with a high percentage of abnormally shaped sperm (morphology) [20]. In severe cases, sperm abnormality may result in total failure of fertilization. Even though the sperm can penetrate zona pellucida to enter the egg, the oocyte still cannot form a pronucleus. Under many situations with normal sperm parameters and good oocytes, about 10–20% oocytes still display 0PN at check fertilization [16]. However, some 0PN oocytes may show parthenogenetical activation and cleave into two or more cell stage, even develop to form blastocysts during their culture [15]. A recent study with 6466 ICSI oocytes has reported that 11.2% of 0PN oocytes formed good-quality blastocysts [16]. Further, the 0PN-derived embryos in conventional IVF cycles also result in live birth [4], and about 11.3% (4966/43,949) 0PN-derived embryos and 275 0PN-derived embryos were transferred in 70 cycles and 13 healthy infants were born with a 17.0% implantation rate [16]. Other studies also respectively reported live birth rate with 0PN-derived embryos as 4.6% (13/285), 23.1% (3/13), 48.1% 913/27), 35.6% (155/435) [15, 16, 17]. These results indicate that some 0PN oocytes may develop normal embryos and transferring them may produce healthy babies. Based on current preimplantation genetic testing (PGT) application, 0PN-derived embryos may be screened to select normal embryo for transfer in the human IVF practice, especially for those patients where no other embryos are available [21].

4. Monopronuclear (1PN) eggs

During fertilization check at 16–18 hours post-insemination, about 2.7–5.5% oocytes following conventional IVF and 4.9–11.4% for ICSI procedure [6] display one pronucleus (1PN). This usually occurs when a defective sperm enters an egg, signaling the egg to form its pronucleus. Meanwhile, the defective sperm is incapable of forming its pronucleus. With only one half of the chromosomes functional, the plan for building an embryo is incomplete. These eggs are known as 1PN and have no potential for life. Embryos displaying only a single pronucleus (1PN) are widely regarded to be abnormal and not suitable for clinical use. It is therefore common practice to discard these embryos following fertilization check. These zygotes with 1PN are thought to be at a higher risk of being haploid, and the transferring these embryos may result in an implantation failure. It is not clear to identify the origin of a monopronucleated egg from either oocyte or sperm. It could be parthenogenetic oocyte activation or an abnormal formation of the nuclear envelope [22]. The latter may result either from the combination of the two genomes into a single PN or from the failure to organize a nuclear envelope around one of the parental genomes. However, 1PN embryos have shown the ability for normal blastocyst development, albeit at a decreased rate, and the ability to produce live births [23]. Recently, several articles about the possible origins of zygotes with 1PN and the chromosomal constitution of the resulting embryos have been published [21, 24]. From these reports, we may assume that a considerable number of embryos originating from 1PN zygotes could have a normal chromosomal constitution, and they could be considered for reproductive purposes in cases where no embryos deriving from normally fertilized zygotes are available. Staessen et al. [25] evaluated 312 1PN zygotes twice (at 16–18 h and at 20–24 h after insemination) and found that 25% zygotes appear the second pronucleus after 4–6 h, which showed why abnormal pronuclear-stage embryos are not necessarily genetically defective [26]. In human IVF practice, 1PN embryos may either be discarded or considered for further culture to observe if they are possibly developing to blastocyst for embryo transfer in case of absence of an adequate normally fertilized embryos (Figure 2).

We found that 1PN embryos with potential formed blastocysts often have normal baby birth. Under some situations, no definitive genetic evidence shows that all 1PN zygotes are chromosomally abnormal. It is possible that some may be chromosomally normal despite the morphologic defects. The 1PN embryos with abnormal chromosomal constitution are very difficult to develop to blastocyst stage. Recent preimplantation genetic testing technology may screen 1PN embryos to determine their chromosome constitution to identify haploid, diploid, or triploid. So far, many papers have reported that some 1PN embryos with normal diploid could develop to blastocysts. These euploid embryos were transferred, and healthy babies have been born [27]. Recently, Usshe et al. have reported that they transferred 175 1PN embryos and obtained 69 live births. Compared with 2PN zygotes, 1PN embryos derived by ICSI have a significantly lower blastocyst development potential and embryo utilization rate (68.3 vs. 32.2%), with a reduced ongoing pregnancy rate (27.3 vs. 28.1%) [21]. Thus, not all 1PN embryos are abnormal and some 1PN embryos are usable. However, we recommend that the 1PN zygotes should be cultured and further observed their embryo cleavage and blastocyst formation until Day 3 to Day 6. Then, these blastocysts will be diagnosed by PGT technique to choose normal chromosome diploid before embryo transfer so that normal healthy babies should be born.

5. Triploidy (3PN)

During the process of IVF insemination, a mature egg is placed with several thousand sperm together. Normally, a mature and healthy egg will allow a single sperm to penetrate oocyte zone pellucida and egg plasma membrane and to enter oocyte cytoplasm. After sperm enters egg, a chemical process immediately occurs within the egg so that oocyte can block any other sperm entering oocyte again to prevent polyspermy fertilization, even though it is surrounded by thousands of sperm. This process is known as the cortical granule reaction. There are two types of mechanisms for polyspermy block: the “zona reaction” and the “oocyte membrane block” to sperm penetration [28]. Some eggs are defective in this process, and the chemical reaction is slow or incomplete, which results in a second sperm or more sperm to enter the oocytes cytoplasm and form a third pronucleus (3PN) or multi-pronucleus (mPN) eggs. This process is known as polyspermic fertilization. Li et al. showed that more retrieved oocytes and higher HCG day peak E2 value could result in 3PN incidence more easily [29]. Abnormal fertilization leading to 3PN formation is a common phenomenon in ICSI and IVF. The 3PN zygotes are observed from time to time after conventional IVF (5 to 8.1%) [13, 30] or intracytoplasmic sperm injection (ICSI, 2.5% to 6.2%) [14, 30]. In early stages of the 3PN zygote development, cellular cleavage is likely to be normal, but their proliferation may arrest because of aneuploidy occurring at a later development stage. However, 3PN embryos are capable of self-correction to make triploid become diploid and form blastocysts [31, 32, 33]. There is a significant difference on self-correction between routine IVF and ICSI derived embryos [31]. Traditional IVF uses more than thousands of sperm, which may result in polyspermic fertilization. These embryos may contain the third set of chromosome. It is very difficult for these 3PN embryos to self-correct or eliminate a set of chromosome. However, the insemination of oocytes by direct ICSI usually eliminates two sperm to oocyte, but it does not prevent in all oocytes the formation of abnormally 3PN fertilized zygotes. These 3PN incidences are generally due to a retention of the second polar body after ICSI, or oocyte activation problem in addition to other factors that have not yet been studied in detail. Recently, Wei et al. have identified the unique genetic mechanisms underlying fertilization failure and suggested artificial oocyte activation technology with ICSI to improve fertilization rate and interfering with or supplementing the relevant genes to improve and restore infertile couple’s fertility [18].

Also, those embryos derived from 3PN by ICSI have more possibility for self-correction [34]. Guar et al. showed that both ICSI-3PN and IVF-3PN embryos are capable of self-correction, but the 3PN embryos derived with ICSI have higher capability of self-correction than the those derived with IVF. The ability of a 3PN embryo to become self-corrected is determined by the parental origin of the extra pronucleus [31]. Another study reported that about 62.5% self-corrected ICSI-derived 3PN embryos could progress back to the blastocyst stage, whereas 54.5% were heteroparental diploid blastocysts [33]. Mutia et al. observed 30 embryos with 3PN zygotes; they found that 33.3% had a normal chromosomal array, with 22 pairs of autosomes and 2 pairs of sex chromosomes. However, during other 66.7% 3PN embryos, triploidy was 43.3%, mosaicism was 13.4%, and aneuploidy was 10%. These studies show that not all morphologically 3PN embryos are genetically abnormal [35].

Based on the above analysis, although most of 3PN embryos are genetically abnormal chromosome composition, a small portion of 3PN embryos may develop to blastocyst stage and these capable development embryos may contain normal chromosome composition. Thus, some reports have showed pregnancy and subsequent delivery of a healthy newborn after the transfer of a blastocyst that developed from a tripronuclear zygotes [36].

In human IVF clinical practice, we strongly suggest that embryos derived from 3PN abnormalities be detected prior to embryo transfer. To obtain euploid embryos, preselection must be done to choose the embryos with the highest implantation potential. Except the current methods of embryo selection such as morphology, metabolomics, and morphokinetics, the most accuracy embryo screen method is preimplantation genetic testing (PGT) or preimplantation genetic screening (PGS). Then, the embryos with normal genetic composition may be selected for embryo transfer.

6. Artificial correction for 3PN embryos by removing one pronucleus

At fertilization checking, if an oocyte shows obviously three pronuclei in the cytoplasm, one pronuclues may be removed by micromanipulation technique. Some studies have attempted to restore the diploid status of 3PN zygotes by microsurgical removal of the extra pronucleus [37, 38, 39, 40]. Some reports showed that the microsurgical removal of a pronucleus from tripronuclear human zygotes has been successful [41]. Further, Kattera et al. reported a birth of a healthy boy after transferring three embryos that developed from microsurgically corrected 3PN zygotes [42]. Our laboratory performed microsurgical removing one PN from 3PN zygotes to artificially correct this abnormal fertilization (Figure 3). The results showed that this procedure could improve the corrected 3PN zygote cleavage and blastocyst formation (corrected zygotes 10.5% vs. intact 3PN zygotes 6.6%), but we did not further examine the chromosome composition from these blastocyst by microsurgical correction, which needs to be further studied. Liao et al. analyzed the chromosomal constitution of microsurgically corrected 3PN embryos and indicated that after correction, the diploid rate of the blastocysts (55.0%) was significantly higher (P < 0.05) than that of the arrested cleavage-stage embryos with non-correction (18.4%). The triploid rate of the microsurgically corrected 3PN zygotes (5.7%) was significantly lower (P < 0.01) than that of intact 3PN zygotes (19.4%) [38].

Figure 3.

Removing one pronucleus from 3PN zygote. A indicates 3PN before removing one pronucleus, and B shows 2PN after microsurgical removing one pronucleus.

Thus, we think that the microsurgical operation can effectively remove one pronucleus from 3PN zygotes. After pronuclear removal, the formed blastocyst may be screened by the current PGT technique to select diploidized blastocysts for embryo transfer in rare embryo patients. Whether these corrected embryos from 3PN zygotes can be used in the clinic practice depends on preimplantation genetic diagnosis and heteroparental identification. Transferring one normal genetic composition blastocyst with removal one pronucleus from 3 PM zygote may obtain the pregnancy similar to normal 2PN formation embryo transfer. However, this microsurgical pronuclear removal often causes some 3PN zygote damage and results in low blastocyst development. Thus, this technique is suited to just some patients without normal embryos to make pregnancy.

7. The application of 3PN cytoplasm

As described above, triploidy can appear either from more than two sperm to enter an oocyte or from retention of the second polar body extrusion because of the oocyte meiotic failure. Oocyte membrane and zona pellucida has a capability to block extra sperm entering oocyte cytoplasm. All these blocks involve a depolarization of the oocyte membrane caused by the influx of Na⁺, and a Ca²⁺ oscillation event activated by the sperm attached to the oocyte membrane. Further, the increased intracellular Ca2+ concentration triggers the exocytosis of cortical granules to release their contents passing the oocyte membrane into the perivitelline space [43]. Thus, 3PN formation has a close concern with egg zona pellucida and membrane and seems not to be associated with egg cytoplasm. This phenomena prompts us to consider the similarity of ooplasm between 3PN and 2PN zygotes [30]. Recently, Kotil et al. have done a detail research on ooplasm difference between 3PN and 2PN zygotes, and they examined actin filament and γ-tubulin localization by immunohistochemistry, evaluated the inner membrane potential of mitochondria by JC-1 staining, and observed oocyte ultrastructure by transmission electron microscopy [44]. Their results showed too much similarity of all ooplasms, especially the mitochondrial membrane potential between 3PN and 2PN zygotes. This finding provides us an idea how to use 3PN zygote ooplasm to correct some advance woman eggs with dysfunction of mitochondria.

In human IVF practice, many aging women still are interested in having their babies from assisted reproductive technology. However, these women often show diminished ovarian reserve symptom, which is associated with reduced quantity and quality of the retrieved oocytes and failure of IVF outcome. For over-40-year-old women, their oocyte quality has a significant reduction. A major reason is due to the lack of enough ooplasm component parts in oocyte cytoplasm, such as synthesis of proteins (microtubules), which supports cytoskeleton organization and mitochondria relocation. The functional competency of organelles is crucial for the quality of oocyte. The mitochondria are responsible for oocyte energy production. Thus, aging women often have low quality of oocyte because of their mitochondria malfunction, which results in aging women’s decreased IVF success rate [44].

In order foraged women to realize their dream to have babies, the only approach is to use donation of eggs, but most aging women do not like to seek for oocyte donation. Thus, we must search for new ways to overcome the problem of aging oocytes in our IVF laboratory. It has been reported that along aging with the decline in oocyte quality, oocyte mitochondrial DNA (mtDNA) shows an obvious defects [45]. The repair of oocyte mtDNA may improve oocyte quality. To date, IVF physicians often give patients the supplement of various compounds, such as antioxidants and coenzymes to improve mitochondrial function and fertility outcome. In the last few years, nuclear transfer has become another alternative treatment approach to improve age women oocyte quality and this tri-parent baby has been born [46]. Germline nuclear transfer is a novel technology on ART that involves the transfer of nuclear genome from an affected oocyte/zygote of the patient to the cytoplast of an enucleated donor oocytes/zygote [47]. Current developed nuclear transfer techniques contain mitochondrial transfer, pronuclear transfer, spindle transfer, blastomere transfer, ooplasmic or cytoplasmic transfer, polar body transfer, and so forth [48, 49].

Nuclear transfer may be performed in either germinal vesicle (GV) stage oocytes [50] or metaphase II stage oocytes [49]. Mature oocytes may be used for micro-spindle transfer [51]. Fertilized eggs may be used for two pronuclear transfer or cytoplasmic replacement. However, all these nuclear transfer procedures need to be performed in third younger partner donor eggs. Using donor eggs is expensive and needs more time based on donor agreement consent. Based on the similarity of ooplasm between 3PN and 2PN zygotes, we may use discarded 3PN zygotes to provide good cytoplasm to replace advance aging women oocyte cytoplasm and help to bring about aging women’s embryo growth.

Ooplasmic transfer is one of the latest innovations in IVF treatment, which involves transferring a small amount of oocyte cytoplasm from normal fertile women into oocytes of women with damaged mitochondria. It has been reported that the children born using this technique will possess cytoplasmic organelles including mitochondria from both their biological mother and donor ooplasm [52]. This is called tri-parents baby who has the mixing of parent and cytoplasmic donor mitochondria. However, it is not easy to obtain donor oocytes for this ameliorated purpose. Recently, Fujimine-Sato et al. have tried to use the cytoplasmic function of abnormally fertilized embryos by pronuclear-stage cytoplasmic transfer to improve oocyte quality [53]. In our clinic practice, we may take out partial cytoplasm of the discard 3PN and inject to the aged woman oocyte to form two kinds of mixed cytoplasm in an embryo (Figure 4). Our primary experience shows that this procedure may improve aging embryo quality. However, this part of ooplasmic transfer does not change aging women’s genetic composition, or inheritable genetic modification. Although an aging woman’s modified embryos still keep her with her husband’s genetic genes in the cell nucleus, but the baby born with this technique might appear epigenetics because the mixed mitochondrial DNA would be passed on to all future generations.

Figure 4.

Advance aging woman oocyte correction. After fertilization, firstly two polar bodies have been removed from normal fertilized zygote. Then, all 3 pronuclei are removed from 3PN zygote and about 30–50% cytoplasm from enucleated 3PN zygote will be taken out and transferred this partial ooplasm into aging woman zygote. After two zygote cytoplasm fusing by electrical purse, one mix embryo of cytoplasm will be formed for further culture. After PGT selection, a normal embryo may be transferred.

Also, 3PN zygote may provide its cytoplasm for nuclear transfer. Yao et al. performed a study about ooplast transfer of triploid pronucleus zygote to reconstruct human-goat embryonic development in which their results showed the 3PN cytoplasmic transfer could significantly improve the early development of humanized new constructed embryos [54]. In human IVF laboratory, we may use discarded 3PN zygote cytoplasm to replace advance aging woman ooplasm to create a new zygote by transfer age woman 2PN into enucleated 3PN cytoplasm (Figure 5). This technology does not use a woman’s coplasm. Although it do not change the woman’s original 2PN genetic constitution, maternal cytoplasm is from a donor. Thus, the donor 3PN cytoplasm must have the effect of epigenetics in this offspring. This technology and outcome on human IVF practice application still need more research in the future.

Figure 5.

2PN nuclear transfer using 3PN zygote cytoplasm. Because the aged woman 2PN embryo has dysfunction of ooplasm, especially their mitochondria malfunction, their 2PN may be transferred to enucleated 3PN cytoplasm so that using 3PN ooplasm may improve aged women’s 2PN normal development.

8. Conclusion

Human IVF laboratory often shows many abnormal fertilization eggs, such as 0PN, 1PN, 3PN, and multi-PN zygotes, and these abnormal eggs are usually not used and typically discarded. Recent research and our observation have showed that further work on these abnormal eggs may provide some hope for advance age infertile couples to realize their desire to have babies in their family. We recommend that 0PN eggs may be done for rescue ICSI or further cultured to next few days to observe their further cleavage and development to blastocyst. Selecting chromosome normal blastocyst for embryo transfer may have healthy baby birth. Many studies showed that not all 1PN embryos are abnormal and some 1PN embryos are usable. The 1PN zygotes should be cultured and further observed their embryo cleavage and blastocyst formation until Day 3 to Day 6. Then, these blastocysts will be diagnosed by PGT technique to choose normal chromosome diploid before embryo transfer so that normal healthy babies should be born. Although most of 3PN embryos are genetically abnormal chromosome composition, a small portion of 3PN embryos may develop to blastocyst stage and these capable development embryos may contain normal chromosome composition. Also, those embryos derived from 3PN, especially by ICSI, have more possibility for self-correction to become normal euploid embryos. The microsurgical removing one PN from 3PN zygotes may artificially correct this abnormal fertilization. After one pronuclear removal, the formed blastocyst may be screened by current PGT technique to select diploidized blastocysts for embryo transfer in rare embryo patients. Some reports have showed pregnancy and subsequent delivery of a healthy newborn after the transfer of the corrected 3PN blastocyst. Because of no obvious difference of ooplasm between normal fertilized 2PN zygotes and 3PN zygotes, this cytoplasm of 3PN zygotes may be used to improve advance age woman poor oocytes and embryo quality. Transferring partial cytoplasm from 3PN to the fertilized 2PN zygote of aged woman may promote her embryo to develop blastocysts. This partial ooplasmic transfer does not change an aging woman’s genetic composition; thus, the aging woman’s embryos still keep her with her husband’s genetic genes in the cell nucleus. However, the baby born with this technique might appear epigenetics because the mixed mitochondrial DNA would be passed on to all future generations.