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Safeguarding medically assisted reproduction

Mulder, C.L.

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Citation for published version (APA):Mulder, C. L. (2018). Safeguarding medically assisted reproduction.

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Download date: 11 May 2020

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General discussion and implications for future research

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General discussion and implications for future research

The main goal of this thesis was to study safety aspects of medically assisted reproduc-tion. Safety may be defined as a non-detrimental effect on the health of the individual undergoing the treatment and its offspring. Ideally, individuals treated for subfertility and their offspring, should be of equal health and disease risk should be of comparable magnitude, compared to non-treated individuals and their naturally conceived off-spring. Of course, medical treatments of subfertility are, as any other medical treatment for other diseases, never fully without risks. If these treatments are offered, counselling of patients before treatment on potential risks of known and/or unknown character should form an integral part of good medical care. Throughout the course of this thesis, we gained a broad vision on the safety of medically assisted reproduction by performing literature studies (chapters 2 and 4), (development of ) hands-on health assessment in a mouse model (chapter 3 and 6), and in-depth epigenetic analysis of placental tissues (chapter 5) to eventually develop an opinion (chapter 6 and this discussion) of this complex matter.

Risks of MAR for patients and their offspring

Risks and consequences of medically assisted reproduction can be studied at several biological levels, from studying small changes in epigenetics to large-scale epide-miological studies. In this thesis, we attempted to gain more knowledge on the health consequences of medically assisted reproduction (MAR) by assessing cancer incidence in recipients undergoing spermatogonial stem cell transplantation (SSCT) in a mouse model (Chapter 3) and studying methylation levels of imprinted genes in IVF and natural conception derived human placental tissue (Chapter 5). In brief, we found no increased risk of malignancies in busulfan treated mice transplanted with in vitro propagated mouse SSCs compared to untransplanted busulfan treated controls (Mulder et al., 2018). Also, we found no statistical significant differences in the mean DNA methylation status of differentially methylated regions (DMRs) associated with parentally imprinted genes in placenta derived from natural conceptions, placenta derived from IVF conceptions cultured in HTF, and placenta derived from IVF conceptions cultured in G5 culture medium.

Regarding SSCT, significant progress has been made towards clinical implementation since initialization of this PhD thesis. A major step in proving the efficacy of SSCT in the future clinic was made when allogeneic transplantation of uncultured SSCs in a non-human primate model was demonstrated to restore fertility (Hermann et al., 2012). Since in vitro propagation is considered crucial for efficient testicular colonisation after transplantation, the genetic and epigenetic stability of cultured human spermatogo-nia was studied as well (Nickkholgh et al., 2014). This study showed no chromosomal

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abnormalities in cultured spermatogonia, however an altered DNA methylation level of some of the selected imprinted genes was identified. Considering safety of SSCT, transplantation of in vitro propagated SSCs in a mouse model was found not to increase cancer incidence in the recipient (Chapter 3, Mulder et al., 2018). Moreover, human testicular cell cultures to which leukaemic cells were added could be efficiently purged from leukaemic cells (Sadri-Ardekani et al., 2014). Practically, cryopreservation protocols for human (prepubertal) testicular tissue were studied and optimized (Baert et al., 2013; Onofre et al., 2016) and an efficient injection technique was developed for the human testis (Faes et al., 2013, 2017). First steps into clinical evaluation of safety of fertility pres-ervation were undertaken as well. A recent study showed that the required testicular biopsy surgery is not harmful for the development of the biopsied testis in prepubertal boys with cancer (Uijldert et al., 2017). Despite the progress that has been made, no systematic evaluation of the long-term health of SSCT-derived offspring in animal stud-ies has taken place yet.

The knowledge on the health of children conceived through MAR in general has increased since the start of this project. A recently published systematic review on the long-term outcomes of IVF and ICSI children raised concerns on neurodevelopment of these children, including a delay in motor and cognitive development, increased risk of neurodevelopmental disorders (e.g. mental retardation, autistic spectrum disorder), although these results could not be reproduced in all studies included in this review (Catford et al., 2017). In the same study, growth and general physical health was found equal between IVF and ICSI children, while IVF/ICSI children had an increased risk of childhood illness and hospital admissions compared to aged matched naturally con-ceived peers.

Of course, IVF or ICSI cannot be viewed as a single environmental stimulus that may lead to transgenerational Developmental Origins of Health and Disease (DOHaD) effects in the offspring. The treatment itself consists of several components that are shown to have an effect on health parameters. Ovarian hyperstimulation was shown to correlate to increased blood pressure in IVF-derived children at the age of 4 years (La Bastide-Van Gemert et al., 2014; Seggers et al., 2014), suggesting that environmental stimuli before fertilization are of crucial importance for health of the offspring. Besides this, it is often debated that health effects seen in IVF/ICSI children are associated with the subfertil-ity of the parents itself rather than with the treatments they underwent to be able to conceive. Time-to-pregnancy, a measure for the severity of subfertility, was indeed associated with suboptimal neurological development in IVF children at the age of 2 years, while ovarian hyperstimulation did not affect the parameters studied in that study (Seggers et al., 2013).

The fact that a common conclusion cannot be distilled from the data on the health of MAR derived children demonstrates that it is extremely difficult to discover the true

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DOHaD-related effects of IVF on the offspring. Besides the fact that results are often hard to interpret due to the observational methodology of these studies and differences in definitions of clinical outcome measures, the treatment itself differs between clinics and changes through time. The fact that even the choice of culture media influences pregnancy rates, birthweight and post-natal weight at the age of two (Kleijkers et al., 2014, 2015, 2016), points out that even relatively small differences can have an effect on the general health of the children. This indicates that heterogeneity in IVF procedures may also cloud the potential adverse effect of assisted reproduction.

The vastness of reports on conflicting data on the safety of IVF and ICSI, techniques both introduced to the clinic without careful preclinical animal studies, stresses the importance of a paradigm shift in which novel MAR need to be tested prior to clinical implementation. But at which stage of development of a novel MAR should safety be tested? And when is a technique considered safe? In this final discussion, thoughts on these issues will be discussed.

How to safeguard health?

“Resulting in healthy offspring” or “the birth of a healthy baby boy/girl” are sentences often seen in publications on novel artificial reproductive techniques (Steptoe and Edwards, 1978; Sato et al., 2011; Kawamura et al., 2013; Tanaka et al., 2016; Jensen et al., 2017; Laronda et al., 2017). By this the authors imply the birth of a few mouse pups or a newborn human child without major (visible) congenital anomalies. However, in most cases minimal information is given on health parameters on only a limited number of generated offspring. This is standard in so-called proof-of-concept papers, and despite minimal health evaluation these techniques are often implemented rapidly in the clinic.

Studying the health of MAR derived offspring seems straightforward, however, health is a state that is extremely difficult to define. The World Health Organisatio states that “Health is a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity”, a definition that was put forward in 1948 and has never been changed since despite criticism (Huber et al., 2011). And also very few people, if any, can meet these requirements. Therefore, a guarantee that a novel MAR results in children of perfect health is utopic, since this is also not the case for those that are naturally conceived. We can only attempt to collect data on possible health risks of novel techniques, and make sure that the health status is not deviant from naturally conceived children, and thereby terming them healthy.

Given the complex molecular regulation of human embryogenesis, occurring at dif-ferent biological levels, it is in fact a miracle that so many children are born healthy. Small perturbations could result in massive deregulation of development, as is elegantly pointed out by effects of IVF culture media (Dumoulin et al., 2010; Nelissen et al., 2012; Vergouw et al., 2012; Zhu et al., 2014; Zandstra et al., 2015; Kleijkers et al., 2016).

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Development is regulated through epigenetics, genetics, transcription, protein assem-bly, endocrine regulation, paracrine regulation and many more processes. These regula-tions all occur simultaneously and are more or less susceptible to environmental cues. This complex regulation makes studying MAR-related effects in human samples, which are also corrupted by (genetic) heterogeneity and confounders, extremely challenging. For example, in this thesis we attempted to provide a molecular reason behind effect of embryo culture medium on the on birth characteristics seen in a recent RCT (Kleijkers et al., 2016). Based on our knowledge and available literature, we reasoned that parentally imprinted genes may play a part in this effect. Hence, in this thesis (chapter 5) we studied the DNA methylation levels of a relatively large selection of parentally imprinted genes in placenta samples (obtained from the foetal site) derived from natural conceptions, IVF conceptions from embryo cultured in HTF culture medium or G5 culture medium. To do so, we sequenced amplicons that were specifically designed to known differentially methylated regions (DMRs) of these imprinted genes. In this study we were unable to detect difference in DNA methylation status in these DMRs in any of the three groups, thereby suggesting that the differences in health outcomes (e.g. birthweight) due to IVF itself or due to the culture medium used is not mediated through the epigenetic state of parentally imprinted genes in the placenta. We suggested to widen views and study the effects of mode of conception and choice of culture media beyond the state of imprinted genes in the placenta. After all, the DNA methylation status of imprinted genes in placenta is only a small part of the regulation of embryogenesis.

Currently our knowledge on early (preimplantation) human embryogenesis is primar-ily based on historical studies (Mall and Meyer, 1921) and in vitro studies where embryo development has now been studied up to day 14 (Deglincerti et al., 2016; Shahbazi et al., 2016). In order to understand the effect that the environment has, we need to learn more about the molecular processes during normal gametogenesis and embryogenesis. Therefore, curiosity driven fundamental studies are of tremendous importance, not only to explain any effects seen in the (pre)clinical stage, but also to spur novel ideas for fertility treatment (figure 1).

Perception of safety and patients’ perspective on novel treatments

“As long as it’s healthy”, is an answer often proclaimed by expectant parents when asked whether they prefer a boy or a girl. This phrase encompasses the need for safe fertility treatments for mother, father and child. After all, no one wishes ill health upon their (future) children.

When couples eventually choose for fertility treatment, it is thought that they base their childbearing decisions on the burden for themselves, effectiveness, (financial) costs and safety of treatment for themselves and their future children (Dancet et al., 2014). Also infertile men that are at their last resort treatment option (TESE-ICSI), put safety of

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the treatment at the highest priority (Hendriks et al., 2016). However, for some couples the craving for a child of their own, to achieve biological parenthood, may eventually lead to concessions. After all, subfertility greatly affects ones psychological and social wellbeing, and the longing for a child can be devastating for both men and women (Zebrack et al., 2004; Mousavi et al., 2013).

Subfertile patients are willing to accept the risk of Ovarian Hyperstimulation Syn-drome for the prospective mother undergoing IVF (Van Weert et al., 2007). Also a risk on multiple pregnancies by transfer of more than one embryo is accepted despite the risks that it harbours for both mother and children (Ryan et al., 2004). In a cohort of women awaiting IVF treatment, when giving the rational discrete choice between a child with physical, cognitive or visual impairment, or no child at all, women even prefer to give birth to a child with a disability than to remain childless (Scotland et al., 2007). This emphasizes that these women are willing to accept a significant health risk for their children to achieve parenthood. Also, a marginal subset of men suffering from non-obstructive azoospermia is willing to accept a risk for congenital anomalies and fertility problems for their children, if it could grant them a child (Hendriks et al., 2014).

When considering the couples’ perspective on risk, one must keep in mind that risk perception is an extremely complicated and personal matter. Human beings in general tend to underestimate their risks, the belief that you are not as vulnerable to threats and risks compared to the average population. Risk perception, and hence the subsequent choices made, are influenced by ones emotions and fears, facts, and trust in profession-als providing these facts (Slovic, 1987; Siegrist, 2000). This is why there is an important role for both scientists and health professionals. Scientists have to provide solid facts about (novel) MARs and health professionals have to provide honest information to couples about their options, and the risks that they may pose for them and their chil-dren, especially when a treatment is still in a clinical trial (figure 1).

The right to have children safely

As in private life, people have to be free to make the choices that they want regard-ing the periconceptional environment of their future children and the risks that they may pose (Fullston et al., 2017). As was defined in the Human Rights in 1966 “… the opportunities to decide the number and spacing of children is a basic human right.” (United Nations Department of Economic and Social Affairs, n.d.). This was elaborated on in the theme Reproductive rights of the World Population Plan of Action a couple of years later. The UN recommends that all countries (1) “Respect and ensure, regardless of their overall demographic goals, the right of persons to determine, in a free, informed and responsible manner, the number and spacing of their children”, (2) “Encourage appropriate education concerning responsible parenthood and make available to persons who so desire advice and the means of achieving it”, (3) “Ensure that family planning, medical and related social

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services aim not only at the prevention of unwanted pregnancies but also at the elimina-tion of involuntary sterility and subfecundity in order that all couples may be permitted to achieve their desired number of children, and that child adoption may be facilitated”. Also, according to the Rights of the Child (1959), the unborn child “needs special safeguards and care, including appropriate legal protection” (Courtesy, 2003). These recommenda-tions were made before artificial reproduction was feasible, but remains highly relevant in the light of medically assisted reproduction.

Now that the techniques that are being investigated in medically assisted reproduc-tion are seemingly getting more complex or invasive (e.g. germ line modifications using CRISPR-Cas9 as the most radical preclinical possibility), we need to ponder on how to balance our reproductive rights against safety of ourselves and our future children. Where to draw the line and say that a reproductive therapy is too dangerous for patients or their children to allow offering such therapy to patients, even if prospective parents are consenting to undergo these therapies to achieve parenthood? Should a new tech-nology be made available to the public, even if there are expected severe side effects, to assure that our procreative liberty (i.e. the freedom to either have children or to avoid

Fundamental research

Preclinical safety studies

Clinical trials

Curiosity driven

Hypothesis generating

Implementation of novel MAR in general healthcare

Idea of novel MAR

Generating informationfor patients

Evaluation and validation

Figure 1. A schematic model for responsible clinically driven research in reproductive medicine. The large arrow indicates the flow through time in which several phases of research need to be conducted. Smaller arrows indicate linkage between the different phases of research. In curiosity driven fundamental research basic laboratory studies are being performed in order to create more insights on the biological and molecular processes that regulate physiological and pathological processes during e.g. gametogen-esis, embryogenesis and pregnancy. At this stage the idea of a novel MAR is born. During preclinical safety studies safety and efficacy are tested in animals or in vitro on human material. During clinical trials the novel MAR is performed in human beings as an experimental therapy. After careful evaluation and validation the novel MAR can be introduced into general health care.

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having them) remains untouched? (Quigley, 2010). When following the recommenda-tions of human reproductive rights, it is in fact an infringement of procreative liberty not to offer it (Quigley, 2010). However, offering MAR that may threaten the health of the child could be considered an infringement of the rights of the child. Moreover, one should keep in mind that research involving pregnant women is only allowed if the research causes minimal risk or minimal burden to the woman and child (Council of Europe, 2014). Should hazardous novel MAR not be implemented, to safeguard and protect the (future) unborn child and its mother?

These brain cracking questions illustrate that there is a need for solid safety and efficacy testing prior to clinical application of novel MARs, as described in this thesis. To eliminate involuntary sterility we need to develop and optimise safe reproductive techniques to safeguard the health of the patient’ and that of their future child, so that patients use these in an informed and responsible manner. At the same time clinicians should be able to give the desired advice needed to gather information from hypothesis driven research on these novel techniques (figure 1).

How to test health consequences of novel MAR preclinically?

Until today, there is no official regulation for the preclinical steps that need to be taken prior implementation of novel MAR, even though the regulation of novel MAR has been discussed extensively by professionals in the field (Schatten, 2002; Dondorp and De Wert, 2011; Harper et al., 2012, 2017; ASRM, 2015). There is an exception for novel MAR techniques that involve the transplantation of cultured cells, e.g. SSCs. These techniques need to adhere to the Advanced Therapy Medicinal Products (ATMP) Law, in which criti-cal characterisation and risk assessment of the therapeutic medical product are part of the approval procedure. In a recently published cross-sectional questionnaire, gynaeco-logists, infertility patients and the general public in the Netherlands expressed a need for regulation for negative consequences of novel fertility treatment, including the risk of (major) congenital anomalies, risk of the child developing chronic disease and the risk of the patient to develop cancer (Hendriks et al., 2018). A need for a pre-set path with the goal to safely introduce the technique into general clinical practice was expressed, as is the case for the standard phases in clinical trials. A well-balanced national or European bioethics committee would be essential in the regulation of clinical implementation of novel MAR techniques (Hendriks et al., 2018).

A paradigm on how to perform research on novel MAR techniques has been proposed earlier (Harper et al., 2012). It involves different phases of basic fundamental research, preclinical research on animals and human embryos, and finally small scale to full scale clinical trials. The preclinical phase should mimic as close as possible the potential novel clinical application; it is an interdisciplinary work method where preclinical safety studies are being executed with the aim to eventually translate the technique in the clinic, while

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keeping an eye on the fundamental data (Figure 1). This phase is important not only to gain knowledge on the safety of the technique, but it is also hypothesis generating.

The preclinical phase includes both in vivo and in vitro studies. Technically, if a novel MAR is proven safe in an in vitro and in vivo animal study one could perform a novel MAR in a small scale clinical setting, in attempt of achieving pregnancy in patients (Harper et al., 2012). But a blueprint on how to perform this preclinical phase is currently lacking. In this thesis, we have expanded the paradigm on how to perform preclinical research using mice (chapter 3 and 6), thereby providing pivotal information on the possible risks on the recipient and offspring of a novel form of MAR. Assessing safety with an in vivo animal model is often considered costly and time-consuming, and the translatability to human can be challenging. After all, despite the low physiological and genetic diver-gence of mice compared to human, no animal model can fully recapitulate the biology of a human being. Therefore in vitro testing of human gametes or embryos created by a novel MAR may be a suitable addition.

In vitro testing of the gametes or embryos provide important information on the fea-sibility and functionality of a novel MAR in human. However, this will provide minimal knowledge on the health consequences for the recipient or offspring. A morphological assessment of the gametes or embryos may provide some information on the gross normality (Veeck, 1988; Balaban et al., 2011). To gain more specific information, next generation sequencing could be performed to study for example epigenetic stability of germ cells or embryos as a form of quality control. But what do these quality scoring measurements really tell us about the health of the offspring? Scientifically speaking, it is extremely interesting to study epigenetics in human embryos in a curiosity driven manner; it would provide tremendous amounts of information on epigenetic regula-tion during embryogenesis (Fig. 1). However, correlation to clinical outcomes, including birthweight, would require a large number of embryos and children to be tested. The molecular mechanisms that may underlie the presumed adaptive effect of a novel technique are therefore difficult to unravel. Translation of in vivo animal data may be challenging, but the translation of data acquired from in vitro human embryos or gam-etes to the health status of a child is even more challenging. Also in vitro testing prior to clinical application in human beings is practically unfeasible for some techniques, e.g. for SSCT in which the prospective father receives a testicular transplantation of cultured spermatogonial stem cells resulting in full spermatogenesis.

Despite these pitfalls, in vitro testing on human embryos is a valid technique to test func-tionality and to some extent efficiency of novel MAR techniques. Ideally, in parallel to in vivo testing in animals, a series of preclinical experiments on embryos created for research using the new MAR tested could contribute to the understanding of the novel technique (Harper et al., 2012) This cannot be achieved on non-viable or viable embryos donated for research, since these embryos have undergone a type of MAR already. However, creating

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embryos specifically for research is currently forbidden in the Netherlands and therefore this type of safety testing is not possible in the Netherlands (Borst-Eilers and Korthals, 2002).

More hurdles on the road

Preclinical safety testing of novel MARs is a costly affair, both in time and refunds. Moreover, this form of research can be considered as not riveting compared to fundamental or clini-cal research. Moreover, as a researcher or clinician, choosing to perform comprehensive preclinical health assessment can be risky, since the researcher might be outrun by others.

The phrase “publish or perish”, as coined in a book by Harold Jefferson Coolidge in 1932, is an expression known to and feared by many researchers (Coolidge and Lord, 1932). In the academic environment, a failure to publish at a frequent basis in high impact journals inevitably leads to low chances in the academic field and problems acquiring research funding (Rawat and Meena, 2014). Similar to the pressure to publish, clinicians and researchers may feel the pressure or urge to race towards clinical implementation, to be “the first” to perform a certain therapy. Being the first to perform a certain treat-ment successfully, in other words a life birth from a novel MAR, gives immense amounts of credit, resulting in higher chances to grants, academic awards etc. This is referred to the rule of priority, the first to report holds the right to the discovery (Strevens, 2003).

Both the concepts of publish or perish and the rule of priority may play a role in why proper safety testing prior to clinical implementation has not been standard in history. The urge to publish first is not only simply out of vanity or pride of the researcher himself, it is a necessity to survive academia. Furthermore, the haste in reproductive technology is pushed even further by profit, competition for private funding or patients demands (Steele et al., 1999; Schatten, 2002; Winston and Hardy, 2002; Van Steirteghem, 2008; Dondorp and De Wert, 2011).

Of course, the academic environment and the paradigms inherit to it cannot easily be changed, however we can still stimulate researchers to perform sufficient pre-clinical research before implementing a novel fertility treatment. The current tendency in the scientific community to give priority to quality of publications rather than quantity is hopeful in this light.

A future where preclinical safety assessment in Medically Assisted Reproduction is standard

Initially, the main goal of this PhD thesis was to assess the risks of SSCT for the recipient and the offspring derived from it. I embarked on this PhD project in 2012 and still being a little naïve, I then reasoned such a preclinical phase was standard in reproductive medicine, and that if SSCT was proven to be “unsafe”, it would not be implemented in the clinic. Throughout the last years and while writing this thesis I became more aware of the dynamics of the field of reproductive medicine that causes this process to be

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less straightforward. Moreover “safety” is mind-puzzling to define. Now, at the end of my PhD thesis hope to refine my initial statements, by including the matters that I have discussed in this last chapter.

I would like to advocate for a change towards a future where preclinical safety assessment in medically assisted reproduction is standard. There is a need for specific studies to systematically investigate consequences of a novel MAR technique prior to clinical implementation in human. Also, more fundamental studies on reproduction and embryogenesis are needed to understand the biological standard and to develop novel ideas to help subfertility patients better in the future. I hope that this thesis raises awareness among researchers, clinicians, publishers, policy makers and funders of (clinical) research on the importance of preclinical health assessment of the future parents undergoing this therapy and their children. This will hopefully in the end lead to a healthier future for the coming generations.

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