Bioethics in human embryology: the double-edged sword of embryo research

Abstract

There has been a significant increase in the use of assisted reproductive therapies (ARTs) over the past several decades, allowing many couples with infertility to conceive. Despite the achievements in this field, a mounting body of evidence concerning the epigenetic risks associated with ART interventions such as ovarian hormonal stimulation, intracytoplasmic sperm injection (ICSI), and in vitro culture (IVC) of oocytes and embryos has also emerged. Induced development of multiple follicles, the IVC media itself, and extended culture may alter the epigenome of both gametes and embryos, resulting in yet to be fully understood developmental, postnatal, and adult life health consequences. Investigators have attempted to decipher the molecular mechanisms mediating ART-induced epigenetic changes using either human samples or animal models with some success. As research in this field continues to expand, the ethical responsibilities of embryologists and researchers have become critically important. Here, we briefly discuss the ethical aspects of ART research, concentrating on the constraints arising from the perceived 'unnaturalness' of many of these procedures. Secondly, we focus on the bioethics and morality of human embryo research in general and how ethically acceptable model systems may be used to mimic early human embryogenesis. Lastly, we review the 14-day culture limit of human embryos and the notion that this rule could be considered of taken into account using new technologies and cues from animal models. The ‘black box’ of early post-implantation embryogenesis might be revealed using embryo models. As long as this distinct moral line has been drawn and closely followed, we should not fear scientific growth in embryo research. Although in vitro fertilization (IVF) is ethically acceptable, research with human embryos to improve its success raises serious ethical concerns that are in need of constant revisiting.

Glossary index: Moral status: the ascription of obligations and rights to embryos on the basis of sentience; Sentience: the capacity of the developing embryo to experience feelings and sensations, such as the awareness of pain; Ectogenesis: the growth of the embryo in an artificial environment outside the mother's body.

Keywords:

• Bioethics
• ectogenesis
• human embryos
• organoids
• animal models
• SHEELFs
• 14-day rule
• extended culture

Introduction

Human in vitro fertilization (IVF), now almost exclusively performed with intracytoplasmic sperm injection (ICSI), involves sperm and oocyte preparation, fertilization, embryo culture, transfer, and/or freezing that are well-established methods for overcoming human infertility. Nevertheless, there is still ethical controversy not only regarding the laboratory methods used to achieve these procedures but also how the manipulation of gametes, insemination approach, prolonged in vitro culture of embryos, vitrification strategies, and invasive pre-implantation genetic screening may threaten the chromosomal and epigenetic integrity of the human embryo. Assisted reproduction promotes the reproductive autonomy of prospective parents on the one hand and exacerbates the potential risks to potential progeny on the other. These biomedical and embryology advances would not have been achieved without the basic research and testing with either human samples or model systems. In fact, ethically acceptable studies of human gametes and failed-to-fertilize oocytes are still commonly performed but limited in scope due to societal regulations. Therefore, synthetic culture systems and additional animal models have been developed to better understand early embryo development and the possible causes of its failure. This research has not been met without challenges from both human rights groups and animal activists. Clinicians, embryologists, and researchers are thus, forced to walk this thin line between advancing human embryo research and strict adherence to specific guidelines proposed by the International Society for Stem Cell Research (ISSCR) Committee (Lovell-Badge et al. 2021).

Infertility is incredibly common, affecting between 8–20% of couples worldwide (Chandra et al. 2013; Sun et al. 2019; ESHRE 2020). There are a number of treatment strategies available to couples. However, one of the greatest technological advances in the field and the most successful treatment option involves IVF. It is estimated that more than 9 million babies have been born worldwide since the birth of the first IVF baby in 1978 (ESHRE 2020). Despite significant advances in the field, the live birth rate from IVF still ranges from 25–50%, depending on maternal age (CDC 2006–2018) and likely other factors that remain to be defined.

There is still significant room for improvement. In addition to the need to increase the success rate, several concerns have also been raised regarding the overall safety of IVF. While the data is often conflicting, both IVF and specifically ICSI, have been associated with increased risks of non-chromosomal birth defects and obstetrical complications (Hansen et al. 2013; Qin et al. 2017; Luke et al. 2021). There are many possible etiologies for these findings, and it is currently unclear whether these increased risks are secondary to the higher rate of multiple gestations, supraphysiologic estrogen levels, culture conditions, gamete and/or embryo manipulation, or simply due to the diagnosis of infertility itself (Luke et al. 2021). Additionally, animal and human IVF studies support the possibility of epigenetically-driven alterations affecting both fetal and placental tissue [recently reviewed in (Mani et al. 2020)]. These alterations have the potential for immediate (i.e., increased incidence of imprinting disorders) and long-term health consequences (i.e., increased risk for metabolic disorders later in life). Overall, the controversy and lack of clarity in the literature emphasize the critical need for additional research to truly understand the impact of this technology.

Bioethics of human gamete research

Although research on human gametes is permitted in most countries to identify molecular pathways and biomarkers of fertility, it is of utmost importance that the quality of the gametes used in research reflects their viability and fecundity as closely as possible to that at the time of collection. However, most human research takes advantage of supernumerary or suboptimal gametes not designated or unsuitable for cryopreservation after an ART cycle. Nevertheless, the aliquot human spermatozoa that remains from the sperm sample used for IVF is often sufficient and reflects the intrinsic high morphological heterogeneity of the original. In contrast, whether a cohort of human oocytes or embryos is of sufficient size to be statistically valid is frequently questioned. For example, out of ten oocytes collected by follicular aspiration, one or two oocytes may be meiotically or cytoplasmically immature, and six or seven are likely to display aneuploidy in cases of advanced maternal age. Moreover, there is a high potential for mixed oocyte quality and maturation heterogeneity within a given oocyte retrieval cycle irrespective of maternal age. Even if the unused oocytes appear morphologically acceptable, their developmental potential may be marginal. This has important implications for the validity of any research performed on human oocytes with variable viability. Moreover, even when many morphologically acceptable oocytes are divided randomly between ART treatment and research, such as when oocyte cryopreservation is not included in the treatment regimen, the odds of having only healthy oocytes in both groups is low. Bearing these constraints in mind, it is evident that the couple's decision to donate human oocytes for research is more complex than the donation of human sperm. It should be noted that sperm quality tends to decrease as paternal age increases. We are just beginning to be aware of the complications associated with older fathers (e.g., autism). However, barring oligozoospermia, the number of spermatozoa in one ejaculate (typically >20 million/ml) largely surpasses the number used for IVF (<10 million/ml), making it more likely that the aliquot of sperm is statistically representative of this heterogeneous sample. Add to this, the ease at which a sperm sample is procured does not compare nor even come close to the hormonal stimulation protocols and surgical procedures that women must endure for oocyte collection. Consequently, it is common for legislative bodies to exempt sperm research from stricter ethical constraints beyond standard written consent and the protection of donor identity. In comparison, the bioethics of human oocyte donation for non-reproductive purposes is another matter entirely, and the literature on this topic is relatively limited. Regulations have not been standardized worldwide. Reflective of a country’s concerns, each has its own rules regarding human oocyte research.

Bioethics of human embryos research - the 14-day rule

The bioethics of research on human embryos is far different from that of human gametes (Hyun et al. 2020; Hyun et al. 2021). Regulation of research using human IVF-derived embryos follows strict and precise guidelines by the prevailing, or rather often legislative view, that life begins at fertilization (Adashi et al. 2020). Even when prevalent religious beliefs extend the onset of life to a few days after fertilization, embryo research is still restricted. From this standpoint, it is almost inconceivable to appropriate human embryos for research in most countries where ART is practiced. It is critical that potential donors clearly understand what their embryos can be used for as part of the informed consent process and whether they will undergo molecular analyses or more controversial manipulations such as cloning and/or stem cell derivation (Kalista et al. 2011; Niemiec and Howard 2020), offering a glimpse into development, after implantation (Tyser et al. 2021). Foremost, it should be clear that only the highest quality embryos will be used for ART. In contrast, surplus, good-quality embryos will be cryopreserved for future use in subsequent cycles and/or allocated to research if not required.

Embryos derived from human IVF are still being used for research in certain countries with minimal controversy despite ethical concerns and legal and religious limitations. In some cases, human embryos were obtained via uterine lavage following in vivo conception to compare quality and chromosomal composition with IVF embryos (Munné et al. 2020). Because the women in this study had to sign away their rights to these embryos to participate, the study garnered criticism for treating the embryos as property and the women as ‘human petri dishes’ (Jones-Nosacek 2020). Some of the first guidelines published on human embryo research stem from the Ethics Advisory Board of the US Department of Health, Education and Welfare (Ethics Advisory Board 1979), which fully supported research on human embryos. This report was in response to the clinical breakthrough of the first IVF baby, Louise Brown, in 1978. The report recommended that embryos should not be kept alive longer than 14 days post-fertilization as this window was viewed as sufficient time to conduct any research on human embryos. The 14-day rule was adopted mainly because, at that time point, embryo development coincides with the formation of the Primitive Streak (PS). This transient structure marks the beginning of gastrulation and, specifically, the formation of the three germ layers, or the ectoderm, mesoderm, and endoderm, that form all the tissues of the fetus. Because the PS typically appears on day 15 of human development, the 14-day rule seemed the most logical and biologically relevant time point for conducting research on human embryos. This proposal was subsequently incorporated into what is known now as the ‘Warnock Report’ in 1984, which was implemented in several countries, including the United States, Australia, Canada, and Japan (Warnock 1984; Hyun et al. 2016), and maintained as an internationally recognized code for scientific research in reproductive medicine. For moral reasons and practical purposes, this rule has been upheld for decades under the assumption that embryo research is undertaken and reported ethically. Indeed, it has led to important findings on infertility, the causes of early miscarriage, and the embryonic origin of congenital abnormalities (Devolder 2015).

As to be expected, ethical issues have been, and continue to be, raised regarding the implementation of the 14-day rule of research on human embryos (Nuffield Council on Bioethics 2017). Some opinions are adamantly against embryo research because proponents hold the belief that life begins at fertilization, as mentioned above. Others argue against embryo research at any stage because of the principle that these embryos might have developed normally and given rise to a person if not 'sacrificed' for science (George and Lee 2009; Jones 2011). More recently, this view was further expanded to include when an embryo might feel pain, and therefore, suffer from the research being conducted. It is thought that the 14-day rule limits the development of the core features necessary for the sensation and awareness of pain, known as key sentience precursors (Castelyn 2020). But there are also strong opinions favoring research on embryos produced by IVF procedures to improve infertility treatment and better understand the fundamentals of human development. Overall, these conflicting views about embryo research are often framed by legislative decision-makers personal and religious beliefs and backgrounds and informed by each country's social, societal, cultural, and religious context before government regulation is reached.

Interestingly, the Warnock report was criticized for considering the public's feelings on human embryo research. According to certain philosophers, not all feelings are moral feelings, but they should be considered for the sake of making a better world and saving lives (Harris 1985). Therefore, the Warnock recommendations were made from an understanding of an ethical point of view, asserting that embryos were human beings until day 14 of development but devoid of the emotional status or cognitive capabilities of human adults.

Research bioethics of extending the 14-day rule - pros and cons

The investigation of human development beyond day 14 is considered a ‘black box’ by scientists (Hurlbut et al. 2017). Following the 14^th day of development and the appearance of the PS, gastrulation occurs, resulting in a primitive gut, the neural tube, and the somites that later become the connective tissues. During this same period, extra-embryonic structures begin to emerge, including the yolk sac and trophoblast that eventually give rise to the placenta. Given that it would be nearly impossible to visualize this crucial post-implantation period in vitro, investigators introduced the first three-dimensional culture systems that enabled human embryo development in the absence of maternal tissues and reignited the need for further research beyond the 14-day rule. Using a combination of cultured human embryos and pluripotent stem cells, these studies elucidated many of the cell fate decisions that mediate self-organization and autonomy of the embryo, as well the success through specific culture media of culturing embryos until the 12^th and 13^th developmental day (Deglincerti et al. 2016; Shahbazi et al. 2016). Additionally, this culture system was used to understand the mechanistic differences in the developmental potential of monosomic versus trisomic embryos and showed that it is dependent on which chromosome is affected (Shahbazi et al. 2020). With this information, we may be able to identify the root causes of implantation failure and miscarriage, potentially improving the long-stagnant success rates of ART procedures. However, the embryos produced in culture do not fully mimic in vivo embryo development and progressively degenerate, suggesting that a more accurate micro-environment is required to support post-implantation in vitro embryo development (Xiang et al. 2020). Nevertheless, following these scientific advances, it has been suggested that the 14-day rule be re-reviewed and possibly extended (Pera et al. 2015; McCully 2021).

Scientific feasibility may justify extending the 14-day rule, but keeping embryo development beyond that limit has raised many ethical, moral, social, and religious issues. The main concerns have been focused on defining an ‘embryo,’ which was expounded as an entity without moral, emotional, or even legal protection. It has been made clear that such definitions are not universal and vary across the world (Hyun et al. 2020), depending on the country and its religions and regulations. An outright ban on embryo research was again rejected, arguing the potential loss of benefits from human research. Nevertheless, extending embryo research beyond the 14^th day of development raises additional concerns regarding personhood. It is believed that upon formation of the three germ layers, an embryo seems to acquire the first grade of sentience that should not be violated. Thus, proponents against extending the 14-day rule argue that embryos may experience pain or suffer during experimentation. However, other studies indicate that even by the 28^th day of development, the embryo might not experience any pain due to the lack of functional neural connections (Hurlbut et al. 2017). In any case, the starting point at which the embryo could experience pain should be taken seriously as the developmental stage at which research should be stopped.

Whether an embryo beyond the 14^th day has the moral status of a person has been the subject of intense debate between scientists and ethicists. As mentioned above, the answer to this question is based mainly on the laws, religions, and regulations of the country where the research takes place. There are distinctions between countries as to when an embryo should have moral status or even acquire legal protection. In the United States, there is the further separation between individual States; for example, the California Institute for Regenerative Medicine (CIRM) specifies that human embryo research should be terminated by day 12 unless prior approval is obtained from an independent oversight committee of citizens. Supporters of the 14-day rule claim that an embryo can split into two up until approximately day 15, resulting in twins. This meant that the embryo was not a unique individual until twinning was no longer possible. Other individuals argue that this reasoning is 'dubious' and suggest that embryo research should not be allowed in principle, regardless of the developmental stage (Blackshaw and Rodger 2021). Other arguments against embryo research are known as the ‘slippery slope,’ or the idea that despite being worthy, experiments require the destruction of human embryos. This has been extended to even if research on embryos is not wrong, it will lead to a slippery slope (Sandel 2004). Additionally, embryo research itself does not directly benefit the embryos being tested but rather future individuals that might have avoided developmental abnormalities by applying the findings from previous studies. Those who view embryo research as a slippery slope do not appear to consider these benefits.

The individuals seeking to expand embryo research must address the broader issue of public trust and confidence. Whether the public (and which segment of public?) will influence the decision to extend or maintain the 14-day rule is a matter of respect and should be taken under the auspices of democratic pluralism (Jasanoff et al. 2019). In a similar framework of respect, the opinions and the ideas of those against embryo research should also be given full consideration and cover all life aspects (Appleby and Bredenoord 2018). It is the responsibility of scientists to uphold moral and ethical standards, while bioethicists should still be motivated by novelty and science. The interface of these two views is likely to gain public confidence and trust so as not to irritate public ‘hordes’ sensitive to moral and ethical issues. For example, if the committee that produced the Warnock report had viewed those that opposed embryo research only as a minority, the prospective extension of research limitations would not have had the majority support, and therefore, would not be accepted. In other words, if the limit to the 14-day rule was simply due to the moral status of the embryo, then it would be difficult to overcome this superimposition of ethical properties and reach a consensus regarding the extension of embryo research. Both views should be welcomed and respected, but where should ethicists draw the line? In accord with the Warnock report, scientists suggested day 14 of development, which was accepted almost by ethicists. Would this happen again if scientists proposed a new line or the ethicists suggested a day of their own? That remains to be determined, and representatives from all social, political, and religious backgrounds should be included in this decision (Jasanoff et al. 2019).

Research bioethics in human cell-based models – new kinds of beings

Because of the 14-day rule, the modern era of human embryo research has reignited the debate with the introduction of embryo models formed from stem cells (Hyun et al. 2020), or Synthetic Human Entities with Embryo-Like Features (SHEELFs) (Aach et al. 2017), as well as the idea of ectogenesis (Baron 2021). While these technological advances do not appear to override the Warnock report per se, they inspire similar, if not identical, ethical reasoning on using these so-called 'synthetic embryos.' Initially, ‘gastruloids’ (Warmflash et al. 2014) appeared to have embryonic features that most closely resembled non-synthetic embryos, indicating that non-synthetic embryos are equivalent to natural human embryos and therefore eliminate the need to use natural human embryos for embryo research. However, they also displayed chronological variability and did not fully mimic early in utero embryonic development. Nevertheless, there is no doubt that the growing field of synthetic biology, combining the abilities of both scientists and engineers to produce increasingly complex tissues, will inevitably lead to the generation of synthetic embryos that exhibit embryo-like features more comparable to their natural counterparts, such as ‘blastoids’ (Kagawa et al. 2022). However, this move forward requires that synthetic embryos achieve totipotency, or the formation of both embryonic and extra-embryonic lineages, potentially countering the 14-day rule. Although not totipotent, human pluripotent stem cells (hPSCs) and appropriate culture media have generated various organoid models for research purposes (Bredenoord et al. 2017). More recently, the identification and isolation of a human trophoblast stem cell population (Okae et al. 2018) has enabled the creation of 3-dimensional embryo models by mixing both embryonic and extra-embryonic stem cells in vitro (Shahbazi et al. 2019; Weatherbee et al. 2021). Further research in this area has also produced expanded pluripotent stem cells (EPSCs) that resemble the epiblast, hypoblast, and trophectoderm lineages (Sozen et al. 2021) as well as yolk sac-like cells (YSLCs) (Mackinlay et al. 2021). Although these structures would normally form before the appearance of the PS, it is only a matter of time before gastrulation-like tissues can be generated based on the progress with animal models (Kyprianou et al. 2020; Lovell-Badge et al. 2021). Thus, the creation of totipotent synthetic embryos might be just the tip of the iceberg regarding the power of bioengineering.

Considering the above scientific advances, it is evident that the 14-day rule could be violated even if these embryo-like entities are not intact embryos because tissues and organs normally formed beyond the 14^th day of embryo development might be generated. It is also possible that synthetic biology and cell engineering approaches may generate SHEELFs with features that do not include the presence of the PS, which is central to the 14-day rule. Even if SHEELFs are engineered to bypass the PS stage, synthetic embryos could still experience pain. This is morally concerning. Cerebral organoids are probably the closest to fit the above description since they can produce brain waves in vitro (Chambers et al. 2012; Jo et al. 2016). Thus, some suggest that SHEELFs have been created as an alternative to natural development, traveling ‘off the beaten path’ (Aach et al. 2017). Research on human embryo entities may fall under previously established legislation and rules, but whether these laws should be enforced ultimately depends on the definition of an embryo. Synthetic embryos are obviously not considered natural (Koplin and Gyngell 2020), but they could resemble naturally conceived human embryos and potentially develop into normal fetuses under the appropriate conditions. For this reason, as recommended by the ISSCR (Lovell-Badge et al. 2021), synthetic embryos should not be transferred into a woman's uterus.

SHEELFs or other embryo models may acquire sentient status or moral-signifying features (Aach J et al. 2017), including the ability to experience pain or pleasure (National Institutes of Health 1994). Therefore, research must not only rely on the appearance of physical features when raising ethical concerns. From a developmental perspective, the ability to experience pain is far past the point where ethical implications should be considered. Since the onset of heartbeat is observed approximately on day 21-23, and the capacity for neural sensation emerges even later, on day 28 of development, accepting this argument favors considering a limited extension of the 14-day rule (Hurlbut et al. 2017). However, the appearance of morally significant features is much less apparent and even if identified in normally developing embryos, whether these characteristics are the same in synthetic embryos is unknown. Suppose the physical but not the moral features are determined to be identical between natural and synthetic embryos. In that case, it could become ethically acceptable to extend research using embryo models, and the ‘black box’ of early post-implantation embryogenesis might be revealed. As long as this distinct moral line has been drawn and closely followed, we should not fear scientific growth in embryo research.

There is also the notion that SHEELFs exhibit moral properties and that their use in research prevents SHEELFs from fully developing them. While it is already difficult to identify completely developed moral features, recognizing partial morality is nearly impossible unless key sentience precursors such as those for the awareness of pain might be used as an indication of morality (Koplin and Gyngell 2020). However, the developmental time points at which moral status signifying features emerge have not yet been identified in the majority of tissues. The initiation of neurulation and detection of neuronal activity are likely the best markers for the termination of embryo research since they are prerequisites to pain receptivity and coincide with the entry of the embryo into morally restricted areas or territories. In addition, since the appearance of a neural network does not necessarily correlate with the experience of pain, a threshold level of pain should also be defined. Upon recognition and acceptance of this threshold, further embryo research beyond the accepted level should be banned, including that in SHEELFs. This offers a conciliatory solution among both opponents and proponents of extending embryo research past the 14-day limit because it respects all appropriate moral boundaries to move forward. Moreover, whether these experiments may result in a new kind of human being should be seriously taken into ethical consideration as Darwin's theory of natural selection could be surpassed. The question of whether science can override or overcome natural selection seems to have been partially answered with the success of IVF itself. Natural selection selects only the fertile couples to conceive. IVF helps infertility couples. Therefore, through IVF, natural selection has been overridden in a way. Accepting that ART is safe enough to be introduced into the clinic, then its implementation for the sole purpose of helping couples to conceive respects ethical boundaries should be re-considered with every new scientific advance. The Dickey-Wicker Amendment is an amendment attached to the appropriations bills for the Departments of Health and Human Services, Labor, and Education each year since 1996 restricting the use of federal funds for creating, destroying, or knowingly injuring human embryos. The Dickey-Wicker amendment only prohibits federal funding for experimentation using human embryos, not the experimentation itself. Although the Dickey-Wicker Amendment is still active, Presidents have pushed the amendment's limits by asking the NIH to allocate funds for additional embryonic stem cell lines. Nevertheless, this amendment prohibited and greatly limited the experimentation on IVF-derived embryos (donated by couples that no longer needed them for IVF procedures), in which many fundamental developmental aspects regarding abortion and infertility would have been solved (Adashi and Cohen 2015). Furthermore, the Dickey-Wicker Amendment dates a long way back, before the advent of techniques that produce embryo-like structures from stem cells. Therefore, scientists working on embryo developmental research need clearer guidance on the type of cells, tissues, and zygotes/embryos eligible for federal funding.

Finally, the idea of ectogenesis, which involves developing an artificial environment for full-term gestation outside the woman's uterus, takes these ethical considerations to another level. Although the prototypical ectogestational technologies have not yet been perfected to support development to term, moral and ethical concerns have already been raised about these strategies. The fact that preterm neonates are supported by incubators using ectogestational technologies may justify this research avenue. Ethical justification for removing a fetus from the uterus and bringing it to term in an artificial environment and the potential risks to a pregnant woman under such a scenario requires careful consideration. Alternatively, perhaps generating an embryo in a 3 D in vitro culture system, followed by an artificial uterus (ex utero), to achieve complete ectogenetic development would be more acceptable given that the artificial womb technologies have already been used as a treatment in medical research (Romanis 2020a). Such an embryo could develop into a ‘gestateling’ with no developmental abnormalities and, ultimately, a healthy neonate (Romanis 2018). However, it is not clear what would happen to gestatelings with non-life-threatening abnormalities and those that perish between the early and late ectogestation periods. These ethical issues cannot be reconciled because gestatelings might display moral properties of sentience and experience suffering and pain. Thus, this technology could be justified in benefiting preterm gestations, but it is hard to imagine that science will reach a level safe enough to conduct research on human fetuses (Romanis et al. 2021). If science reaches this tipping point, gestatelings will have moral status and likely legal protection even if they have been created for research. One of the most significant ethical issues will be the termination threshold, which in most countries is earlier than two-thirds of gestation, raising the question of whether a gestateling should be allowed to continue in cases when abnormal development occurs. Considering the advances in fetal therapy that have rapidly moved from use in animals to humans, it may be possible to repair developmental abnormalities, whether life-threatening or not. Nonetheless, the prospect of full ectogenesis for the creation of human beings will be hotly debated by embryo researchers and ethicists for years to come regardless of its attainability (Romanis 2020b; Baron 2021).

Bioethics of other scientific advances – mitochondrial replacement and gene editing

Maternal Spindle Transfer (MST), or the technique of transferring the genetic material of a woman with an inherited mitochondrial disease into the cytoplasm of a donor oocyte, was initially used for mitochondrial replacement therapy (MRT) to avoid passing on mutated or otherwise damaged mitochondrial DNA to her offspring (Kang et al. Nature 2016). More recently, this strategy has also been assessed for its ability to improve embryo development after fertilization in ART patients by replacing potentially damaged or aged maternal mitochondria with young, healthy mitochondria of oocyte donors. Although MST and research on embryo models share similar ethical issues (Bredenoord and Appleby 2017), MST invokes another ethical debate. MST can produce a heteroplasmic child with the genetic makeup of three parents, the mitochondrial DNA of the ooplast donor, and the nuclear DNA of the ART patient-parents; hence, it's often referred to as 'three-person IVF.' Efforts to explore human germline modifications for the conception of genetically modified children with a decreased risk for developing transgenerational mitochondrial diseases (Newson et al. 2016) are viewed as eugenics by some. Several articles have been published regarding the ethical issues of eugenics based on such techniques (Wrigley et al. 2015; Rulli 2016). With the motivation that future generations could be free of genetic diseases, research on embryo models in the 3 D culture systems may also result in the production of eugenic children. This borders on the extension of embryo research beyond the 14-day.

New ethical challenges have also emerged with the advent and widespread use of CRISPR-Cas9 gene editing and its potential application to human embryos and fetuses. Notwithstanding the ethical implications of gene editing itself and the potential for off-target effects, the impact of this technology on the gametes raises significant moral and legal issues. It can affect the next generation and beyond through germline transmission (Beriain 2017; Cohen and Adashi 2021). Gene modification of gametes to produce embryos promotes another state of eugenics, which to some seems that this is not so much a matter of ethics, but a matter of law (Cohen et al. 2020). Public opinion regarding gene-editing technologies should be solicited and general approval sought since such decisions can influence the entire human population (Adashi et al. 2020). Based on how far we have come with human-assisted reproduction, it seems likely that additional scientific advances and experimental technologies that we have not yet imagined will emerge in the next several years for ethical consideration (Adashi and Cohen 2020).

Research bioethics in the embryology of animal models

There has been a fairly long history of combining human male gametes with surrogate female gametes from animals for fertility testing. This includes the hamster zona-pellucida free egg penetration assay to test human sperm fertilizing ability. More recently, the sperm centrosome function assay in which patients' live spermatozoa are injected into other mammalian oocytes or de-membranated human spermatozoa are co-incubated with frog egg extracts has been considered (Song et al. 2021). While obvious to developmental biologists, the public may miss the fact that such inter-specific gamete mixing does not lead to the formation of viable embryos. In extreme cases, this could be interpreted by a lay person as an attempt to create a human-animal chimera, and must be considered, as it has been accomplished experimentally (Tan et al. 2021). As naïve and harmless as this notion may seem, creating morally ambiguous entities (Subbaraman 2021) could turn public opinion against meaningful research on human gametes, thus hindering further optimization of human ART. However, the perception and misunderstanding of recent research on inter-species synthetic embryos are more concerning. The most provocative of which is the rhesus macaque-human embryo model (Subbaraman 2021). This is likely because non-human primates such as the rhesus monkey are genetically similar to humans and share evolutionary closeness on the phylogenetic tree. Because not every aspect of research can be modeled in silico and the generation of human embryos for non-reproductive purposes is prohibited in most countries, the continued use of other species to test human fertility and/or improve IVF success is inevitable until the study of human embryology in vitro or ex vivo is accepted beyond the 14-day rule.

Future directions & conclusions

The future of embryology is based on tools that combine biology, physics, engineering, and artificial intelligence (AI) (Brivanlou et al. 2021; Brivanlou and Gleicher 2021). Early embryogenesis is associated with massive transcriptional changes and global epigenetic reprogramming. Therefore, research requires systems biology that takes a multi-omics view to understand embryology thoroughly, and due to the 14-day rule, ‘comparative embryology’ to unravel the mechanisms of human embryo development. Recent advances include single-cell multi-omics studies on early human embryos, which enabled dissecting the complex and highly coordinated epigenetic reprogramming during human pre-implantation development (Li et al. 2018) and has been explored from spermatogonia/oocytes to blastocyst (Estill et al. 2019). Moreover, a single-cell multi-omics study of chromatin accessibility, DNA methylation, and RNA expression during the onset of gastrulation has been performed in mouse embryos (Argelaguet et al. 2019). The challenge in this field (as in many others) is bringing together the large quantity of data from various organisms scattered across various publications and data repositories to address these fundamental questions. Understanding the molecular mechanisms underlying human embryology would benefit from a well-coordinated integrative project combining published data from various sources, knowledge transfer from different animal models, and results of synthetic human embryology. These efforts would begin to close our gap in knowledge on human embryos beyond 14-days and complement existing data on early embryonic development. A convergence between embryology and modern genomics tools is expected in the near future, rapidly advancing various fields such as ‘molecular embryology’ and ‘systems embryology.’

The framework we provide here is meant to stimulate discussion rather than advocate for or against extending embryo research. Ectogenesis is not a matter of ‘if’ but ‘when.’ Political and religious considerations aside, it is difficult to prevent or universally ban such research. In his 1865 science fiction novel From the Earth to the Moon, Jules Verne foresaw interplanetary travel. Aldous Huxley's 1932 dystopia Brave New World famously predicted that test-tube babies would populate the future world. We have now put human footprints on the Moon, and over 9 million of those test tube babies are helping to populate Earth. We should take the preferred path that embarks on having these difficult discussions about the near future of human embryo research now, rather than to assume that ‘it’ will never happen.

Authors' contributions

Conceptualization: GA, PS, SAK; writing—original draft preparation: GA, PS, PJT, SLC, TK, CIM, SBS, AM; writing—review and editing: GA, PS, EYA, SAK; supervision: SAK. All authors have read and agreed to the version of the manuscript.

Abbreviations
ART	=	assisted reproductive therapy
Cas9	=	CRISP-associated protein 9
CRISPR	=	clustered regularly interspaced short palindromic repeats
hPSCs	=	human pluripotent stems cells
ICSI	=	intracytoplasmic sperm injection
ISSCR	=	International Society of Stem Cell Research
IVC	=	in vitro culture
IVF	=	in vitro fertilization
MRT	=	mitochondrial replacement therapy
MST	=	maternal spindle transfer
NIH	=	National Institute of Health
OPU	=	ovum pick up
PS	=	primitive streak
SHEELF	=	synthetic human entities with embryo-like features.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.