In vitro fertilisation/intracytoplasmic sperm injection beyond 2020

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1 DOI: / Review article In vitro fertilisation/intracytoplasmic sperm injection beyond 2020 GE Crawford, a,b WL Ledger a,b a The Royal Hospital for Women, Randwick, NSW, Australia b School of Women s and Children s Health, The University of New South Wales, Randwick, NSW, Australia Correspondence: GE Crawford, The Royal Hospital for Women, Barker Street, Randwick, NSW 2031, Australia. giselle.crawford@health.nsw.gov.au Accepted 22 October Over 8 million babies have been born following IVF (in vitro fertilisation) and other artificial reproductive technology (ART) procedures since Louise Brown s birth 40 years ago. New innovations have added much complexity to both clinical and laboratory procedures over the last four decades. Translation of novel approaches from basic science into clinical practice continues unabated, widening the applicability of ART to new groups of people and helping improve both chances of healthy live birth and patient acceptability. However, the impact of ART on the health of both patients and their offspring continues to cause concern, and many ethical challenges created by new scientific developments in this field attract widely differing opinions. What is undeniable is that there will be a sustained global growth in utilisation of ART and that reproductive tourism will allow many people to access the treatment they desire notwithstanding national regulations that may forbid some approaches. The greatest challenge is to expand access to ART to those living in the less wealthy nations who are equally deserving of its benefits. Keywords Assisted conception, basic science, infertility. Please cite this paper as: Crawford GE, Ledger WL. In vitro fertilisation/intracytoplasmic sperm injection beyond BJOG 2019;126: Introduction Over 8 million babies have been born following IVF (in vitro fertilisation) and other artificial reproductive technology (ART) procedures since Louise Brown s birth 40 years ago. 1 Several monumental scientific advances have been made in the world of ART over the past four decades. The 1980s saw significant improvements in the technology of what we might now refer to as traditional IVF, the 1990s heralded the introduction of intracytoplasmic sperm injection (ICSI) and the new millennium saw the rise of vitrification and genetic testing of the embryo. Once only a treatment for tubal factor infertility, IVF and its related technologies are now a means to pregnancy and a family for patients faced with almost all causes of infertility. This paper will consider the exciting new developments and potential future directions in ART, and in doing so will predict what IVF/ICSI might look like beyond Growth in the sector The global trend of delayed childbearing, steady and sustained increase in IVF success rates, and rise in disposable income have all contributed to the ongoing market growth in global IVF services. Worldwide, the IVF services market generated $10.5 billion in revenue in 2017 with projections for it to continue growing at compound annual growth rate of 9.8%, reaching $22.5 billion by Global ART registry reporting data have confirmed that the number of ART cycles continues to increase; however, utilisation remains under the influence of affordable access to ART, with regional differences persisting. 1 There is no sign of market saturation, with new markets opening up notably in Asia-Pacific (APAC), where the demand for IVF treatment is expected to boom owing to fertility tourism, increasing foreign industry interest, and initiatives such as ASPIRE (The Asia Pacific Initiative on Reproduction) promoting awareness and therefore growth. IVF for the non-infertile There is an increasing demand for IVF from healthy fertile couples wishing to prevent the transmission of genetic disease. This has been spurred on by the combination of wide availability of pre-conception carrier screening (PCS), vast increases in the number of known identifiable genetic diseases, and a reduction in costs associated with human genome sequencing. PCS enables the detection of carrier status 237

2 Crawford and Ledger of recessive disease(s) in individuals who do not have a known increased risk of being a carrier on the basis of their family history, thereby identifying couples with an increased risk of having an affected child allowing informed reproductive decision making. 3 Pre-conception carrier screening has traditionally only being available to couples at risk for a particular disease, the classic example being Ashkenazi Jews, who often undergo screening for the mutated HEXA gene linked with Tay-Sachs disease, a fatal genetic disorder. With advances in genomic sequencing and the availability of expanded carrier screening testing an individual s carrier state for multiple conditions at once, regardless of ethnicity, it is now technically possible to screen everyone to determine their carrier status. It has recently been recommended by the American College of Obstetricians and Gynecologists that carrier screening be offered to all patients considering pregnancy or already pregnant. 3 With PCS including expanded carrier screening becoming an increasingly utilised gateway into IVF, beyond 2020 we may see an exponential demand for IVF with preimplantation genetic testing (PGD-T). Concern is often raised by the general public and media alike about such procreative liberty leading to the advent of designer babies, with couples selecting embryos on the basis of favourable traits; however, the Human Fertilisation and Embryology Authority (HFEA) Code of Practice 4 remains clear that an embryo may only be tested if the particular chromosomal abnormality in question may affect its capacity to result in a live birth, or where the abnormality might result in a serious medical condition. It can be debated whether a serious medical condition includes adult onset disorders such as Huntington s disease and BRCA mutations. Sex selection for non-medical indications is commonly practised in some parts of the world such as the USA, 5 while not allowed in others countries such as the UK and many European countries. This is contributing to the emerging dilemma of couples seeking cross border reproductive care that, beyond 2020, requires the establishment of reliable national and global registries to avoid the potential for widespread harm. Elective oocyte-freezing for fertility preservation is now a well established component of ART and provides a viable option for both medical and social reasons. For many women it is not cancer but simply the passage of time that renders them infertile, with women often delaying their childbearing due to social, educational, and financial pressures. The option of oocyte donation is often very limited due to limited supply, ethical, and legal concerns, and women resoundingly prefer to use their own genetic material where possible. In 2013, the American Society for Reproductive Medicine (ASRM) declassified oocyte-freezing as experimental following the publication of trials demonstrating similar pregnancy rates from IVF with vitrified/warmed oocytes compared with fresh oocytes, spurred on by the development of vitrification systems. 3,6,7 While often referred to as an insurance policy, there is concern about the false sense of security that social egg-freezing can create, given that even large numbers of cryopreserved eggs cannot guarantee a baby in the future. However, with its increasing acceptance and accessibility, beyond 2020, demand for oocyte cryopreservation will continue to increase both for those afflicted with cancer and other medical conditions, and for the extension of female fertility. New technologies Not only do emerging technologies hold much promise for improved outcomes in ART, they may also provide a means for greater efficiency and acceptability. Much research continues on varied approaches to selecting the best embryo for transfer. The purpose of PGT-A is to facilitate the selection of embryos with the highest likelihood of leading to live birth, to facilitate single embryo transfer (SET), and to reduce the risk of chromosomal abnormalities in the resultant child. 8 The currently utilised day 5 multi-cell trophectoderm biopsy is thought to have a much improved embryo implantation rate and thereby has replaced the old day 3 blastomere biopsy; additionally, with the implementation of next generation sequencing (NGS), a higher level of accuracy and lower cost can be achieved. 9,10 After 10 years of rapidly increasing utilisation of pre-genetic testing for aneuploidy (PGT-A), its use has come under the spotlight recently due to the complexity in defining and determining the fate of a mosaic embryo, potentially resulting in the discarding of potentially viable embryos Mosaicism is thought to arise from mitotic cell divisions after fertilisation, and mosaic cells may reside within the inner cell mass, destined to become the fetus; within the trophectoderm, destined to become the placenta; or in both. Recent studies using NGS have demonstrated that mosaic embryos can implant and become healthy babies and, conversely, embryos previously diagnosed as euploid have been shown to be mosaic. 11,15 This recent controversy has cast doubt onto the value of PGT-A, rendering it unsuitable as a routine screening diagnostic tool, and subsequently its utilisation is likely to decline in IVF/ ICSI beyond 2020, with some calling for a ban on its use outside of clinical studies. Many non-invasive techniques for selecting the best embryo have been developed to avoid the invasive embryo biopsy that is necessary for PGT-A. With limited evidence that well studied tools such as time-lapse morphokinetics and metabolomics of spent embryo media improve clinical outcomes, focus is now moving to cell-free DNA (cf-dna) for aneuploidy screening in embryos Despite greater accuracy with cf-dna from spent culture media (SCM) 238

3 IVF/ICSI Beyond 2020 after day 5 than after day 3, the ploidy concordance rate of cf-dna of SCM compared with that of trophectoderm biopsy is limited, reported as 65% by Ho et al. 19 and 84% by Xu et al. 20 It is possible that maternal contamination from the cumulus complex cells and mosaicism might contribute to diagnostic inaccuracy. 19,21 More promisingly, comparison between cf-dna from blastocoel fluid (BF) aspirated from blastocysts and trophectoderm biopsy reveals a very high ploidy concordance of 97%, 22 although other groups have not reported such high figures. In many instances, BF removal is a routine step performed to reduce the risk of crystal formation for blastocyst cryopreservation, and appears to have no detrimental effect. While there are still many factors to be considered and further work to be done before wide implementation, non-invasive PGT-A by cf-dna holds great promise for the future. Freeze-all cycles GnRH antagonist pituitary down-regulation protocols are increasingly becoming the mainstay in IVF practice, primarily due to the benefits of reduced ovarian hyperstimulation syndrome (OHSS), which can be further reduced by a GnRH agonist trigger in place of the traditional hcg trigger. 23 However, GnRH triggering leads to a drastic luteolysis and luteal phase defect secondary to negative steroid feedback, resulting in suppressed pituitary LH release. Agonist triggering aside, much concern has arisen regarding the adverse effect on the endometrium of controlled ovarian stimulation (COS) due to the impact on endometrial receptivity. 24 Frozen thawed embryo transfer in a subsequent cycle can circumvent this issue, maintaining increased safety while demonstrating improved pregnancy rates and better obstetric and perinatal outcomes, albeit with some limitations as to the quality of evidence. 24,25 In particular, OHSS cases on the moderate to severe end of the spectrum should no longer occur. This goal can be achieved with use of an antagonist protocol, agonist trigger, use of a freeze-all strategy, and transfer of the embryo in a subsequent frozen thawed cycle, while maintaining high cumulative live birth rates. 26 Mild stimulation regimes The concept of mild ovarian stimulation returns to the original aim of IVF as an attempt to imitate nature. Although conventional high dose gonadotropin ovarian stimulation with multiple embryo transfers has greatly improved the chances of pregnancy and live birth that can be achieved with IVF, this has been at a cost of significant morbidity and even mortality associated with complications including OHSS and multiple pregnancy. Progressive improvements in embryology now allow for good pregnancy outcomes after using milder stimulation regimes with deliberately lower oocyte yields. Three randomised controlled trials in women considered to be poor responders to ovarian stimulation have shown similar pregnancy rates with mild ovarian stimulation versus the standard high-dose stimulation regimens, contradicting the concept that high FSH doses might improve the outcome for such patients. Similarly, a recent randomised controlled trial assessing women with predicted hyper-response undergoing IVF/ICSI with a reduced FSH dose (100 IU/ day) compared with standard dose (150 IU/day), resulted in similar cumulative live birth rates and, importantly, lower rates of OHSS, although not of severe OHSS. 27 Such results question the validity of traditional high-dose stimulation regimes and strengthen the call for mild stimulation for all or most patients. In vitro maturation In vitro maturation (IVM) is emerging as a viable alternative to the conventional hormone-driven ART regime. This technique involves collecting immature oocytes from small antral follicles, with the final stages of meiosis completed in vitro. 30 The major advantage of IVM is that as a minimal approach IVF, it requires less or no gonadotropin stimulation, thereby eliminating the risk of OHSS, notably for the predicted hyper-responder who would otherwise be at increased risk. 31,32 IVM may also be a valuable tool in the onco-fertility setting for both transvaginally aspirated immature oocytes and for enhancing the fertility potential of cryopreserved ovarian tissue. 33,34 The main limitation of IVM to date has been its efficiency compared with conventional IVF. With improved IVM protocols and optimisation of IVM culture systems, success rates have improved, with reported meiotic maturation rates per cumulus-oocyte complex (COC) of up to 70% and clinical pregnancy rates per embryo transfer of 35 43%. 31,35,36 As is often the case with newly emerging IVF technologies, safety data are limited, but early reports on both the obstetric outcomes of pregnancies following IVM and the resulting health of the children are promising Intracytoplasmic sperm injection (ICSI) Over 25 years since the birth of the first child born via ICSI, there continues to be much debate about its expanding use and with ongoing safety concerns. 41 Its initial introduction helped to revolutionise fertility treatment for men with severe male factor infertility who would otherwise never greet their biological offspring. Controversially, indications for ICSI now encompass treatment of mild male factor infertility, unexplained infertility, fertilisation failures, and mixed male/female infertility. ICSI is now used in approximately two-thirds of fresh ART cycles compared with conventional IVF accounting for one-third, with the ICSI:IVF ratio varying from 1:4 in Asia to an astounding 60:3 in the Middle East. 42 An increased risk of adverse 239

4 Crawford and Ledger birth outcomes from ICSI compared with those seen in IVF with fresh embryo transfers has been demonstrated; however, embryo cryopreservation and frozen thawed embryo transfer appear to be protective. 43 It remains unclear whether major confounders are at play, including the possibility of transmission of genetic abnormalities causing severe male factor infertility in the offspring. Given the current widespread use of ICSI for weak indications and ongoing concerns about its safety, we may see a reduction in the utilisation of ICSI as we move forward. Advances in endometrial biology The endometrium, the maternal biological interface dedicated to interactions with the embryo and the feto-placental unit, is a critical tissue for normal fertility and reproductive outcome. 44 Embryo implantation only occurs following complex synchronisation between the blastocyst and endometrium, during the window of implantation (WOI), a time-critical stage lasting up to 4 days in the mid-luteal phase. Previous methods for identifying the WOI via endometrial histological dating have now been replaced by newer technologies involving a variety of omics, including genomics, transcriptomics, proteonomics, and metabolomics. These new tools have revived interest in the study of the endometrial factor, particularly for patients with recurrent implantation failure (RIF). Endometrial receptivity array (ERA) utilises microarray molecular analysis for the expression of multiple genes that interact with endometrial receptivity and implantation. An endometrial biopsy is collected in the mid-luteal phase with results denoted as pre-receptive, receptive or post-receptive, with as many as one in four patients having a nonreceptive endometrium, allowing adjustment of the timing of embryo transfer in a subsequent cycle. 45 While this test is currently on offer commercially, there remains limited evidence of benefit for improving live birth both in good prognosis patients and in patients with RIF The older patient With the rapid decline in fecundity seen with advancing female age, coupled with the societal changes in preferred age for child-bearing, fertility is primarily becoming a disease of ageing. 48 Unlocking the means to improve the fertility potential of the ageing oocyte is the next frontier for 21st century ART and for developed societies. This area of research has a large ethical dimension and there is much debate about the wisdom of accepting pregnancy for women beyond the extreme limits of natural conception at ages above 50 years. Methods to improve oocyte health for older women address both nuclear DNA, using approaches such as treatment with antioxidants such as coenzyme Q10 49 and androgens such as DHEA, and cytoplasmic health, using mitochondrial transfer 50 or transfer of cytoplasm taken from oocytes from younger women. 51 Although there is much excitement about the potential for prolonging the reproductive lifespan of women using such approaches, there is as yet little evidence of progress and there have been several false dawns thus far, with ongoing concerns over the safety of these techniques. 52 New diagnostics The oocyte-secreted growth factors, growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15), are oocyte-specific factors and have important roles in the regulation of oocyte competence and oocytecumulus interaction. As such these molecules may be useful serum biomarkers of female reproductive function. GDF9 and BMP15 are both members of the transforming growth factor-b (TGF-B) superfamily. BMP15 promotes folliculogenesis-stimulating granulosa cell growth, with mutations in the BMP15 gene associated with premature ovarian insufficiency (POI) GDF9 is also expressed exclusively in the oocyte and acts in a synergistic manner with BMP15 on surrounding granulosa cells. 55 It has been shown recently that GDF9 and BMP15 are detectable in female serum and GDF9 concentration correlates with the number of oocytes retrieved during IVF. 56 These factors point towards the future use of GDF9 and BMP15 as diagnostic and predictive serum biomarkers, for use alongside or in preference to anti-m ullerian hormone (AMH). 56,57 Low-cost options and equality of access for the developing world Infertility is not a disease confined to those living in the developed world, with an estimated worldwide median prevalence of 9%, 58 yet there remains a dichotomy of access to fertility treatment between developed and lessdeveloped nations. The psychosocial and economic consequences of infertility for couples, and notably women, in developing countries are often more profound than those seen in Western societies. 59 For many couples, infertility is more than a health problem, it is a social, public health and human rights concern, as all men and women who desire to have children should have the opportunity to do so Low-cost IVF focuses on affordable stimulation protocols, clinical judgment rather than excessive investigations, limited use of disposable materials, and clear and simple laboratory protocols, all of which ensure that low cost does not jeopardise quality. 62 An option for affordable stimulation protocols involves use of cheaper biosimilar FSH preparations that are now emerging as patents expire on innovator products, although questions remain regarding their efficacy and safety profile Other low-cost protocols such as natural cycle IVF, low stimulation regimens, 240

5 IVF/ICSI Beyond 2020 use of oral ovarian stimulants such as clomiphene citrate, and single embryo transfer (SET) can not obviate the necessity for a quality good laboratory Possibly in the future we will see further innovations to help overcome high laboratory costs, such as advances on prior work using the vaginal cavity of the patient as a substitute embryo incubator, with reported single live birth rates of up to 24%. 69 Infertility treatment in low-resource settings is a challenge for policy-makers, and beyond 2020 we must not lose sight of the basic fundamentals of human rights in relation to access to fertility treatment Further roll out of low cost initiatives is essential in achieving equity of access to fertility treatment for less-developed nations and in helping millions of couples to achieve their desire to have a biological child. Conclusion Artificial reproductive technology will continue at the frontier of scientific research in years to come, reaching beyond the limits of our current imagination, while continuing to challenge contemporary ethical concepts. While cutting edge scientific discoveries offer great excitement and hope for the future, we should maintain a continued focus on improved safety and patient acceptability, and aim for further progress in global accessibility. The millions of children born via this technology to date are a testament to the enduring desire for individuals and couples to have a family. This basic human drive is unlikely to dissipate beyond Disclosure of interests GC declares no competing interests. WL declares no competing interests. Completed disclosure of interest forms are available to view online as supporting information. Contribution to authorship Both authors contributed to the concepts and preparation of the manuscript, and read and reviewed the final manuscript. 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7 IVF/ICSI Beyond Chand AL, Ponnampalam AP, Harris SE, Winship IM, Shelling AN. Mutational analysis of BMP15 and GDF9 as candidate genes for premature ovarian failure. Fertil Steril 2006;86: Mottershead DG, Sugimura S, Al-Musawi SL, et al. Cumulin, an oocyte-secreted heterodimer of the transforming growth factor-beta family, is a potent activator of granulosa cells and improves oocyte quality. J Biol Chem 2015;290: Kumar R, Alwani M, Kosta S, Kaur R, Agarwal S. BMP15 and GDF9 gene mutations in premature ovarian failure. J Reprod Infertil 2017;18: Riepsaman A, Chan K, Sweeten P, Somerville E, Walker G, Garrett D, et al. Oocyte-Secreted Serum Biomarkers and Reproductive Potential in Women. FSA 2018 conference proceedings; Sep 9-12, 2018: Melbourne, Australia. 57 Persani L, Rossetti R, Di Pasquale E, Cacciatore C, Fabre S. The fundamental role of bone morphogenetic protein 15 in ovarian function and its involvement in female fertility disorders. Hum Reprod Update 2014;20: Boivin J, Bunting L, Collins AJ, Nygren GK. International estimates of infertility prevalence and infertility treatment seeking: potential need and demand for infertility medical care. Hum Reprod 2007;22: Ombelet W, Goossens J. Global reproductive health Why do we persist in neglecting the undeniable problem of childlessness in resource-poor countries? Facts Views Vis Obgyn 2017;9: United Nations. Report of the International Conference on Population and Development, Cairo, Egypt, 5 13 September New York: United Nations Population Fund, E.95.XIII.18; World Health Organization Department of Reproductive Health and Research. Reproductive Health Strategy to Accelerate Progress Towards the Attainment of International Development Goals and Targets. Geneva: WHO; 2004:WHO/RHR/ Bahamondes L, Makuch MY. Infertility care and the introduction of new reproductive technologies in poor resource settings. Reprod Biol Endocrinol 2014;12: Orvieto R, Seifer DB. Biosimilar FSH preparations are they identical twins or just siblings? Reprod Biol Endocrinol 2016;14: Mastrangeli R, Satwekar A, Cutillo F, Ciampolillo C, Palinsky W, Longobardi S. In-vivo biological activity and glycosylation analysis of a biosimilar recombinant human follicle-stimulating hormone product (Bemfola) compared with its reference medicinal product (GONAL-f). PLoS ONE 2017;12:e Patki A, Bavishi H, Kumari C, Kamraj J, Venugopal M, Kunjimoideen KU, et al. Urinary versus recombinant gonadotropins for ovarian stimulation in women undergoing treatment with Assisted Reproductive Technology. J Hum Reprod Sci 2018;11: Ferraretti AP, Gianaroli L, Magli MC, Devroey P. Mild ovarian stimulation with clomiphene citrate launch is a realistic option for in vitro fertilization. Fertil Steril 2015;104: Van Blerkom J, Ombelet W, Klerkx E, Janssen M, Dhont N, Nargund G, et al. First births with a simplified culture system for clinical IVF and embryo transfer. Reprod Biomed Online 2014;28: Ombelet W. The Walking Egg Project: universal access to infertility care from dream to reality. Facts Views Vis Obgyn 2013;5: Lucena E, Saa AM, Navarro DE, Pulido C, Lombana O, Moran A. INVO procedure: minimally invasive IVF as an alternative treatment option for infertile couples. ScientificWorldJournal 2012;2012: Ombelet W. Reproductive healthcare systems should include accessible infertility diagnosis and treatment: an important challenge for resource poor countries. Int J Gynaecol Obstet 2009;106: Hovatta O, Cooke I. Cost-effective approaches to in vitro fertilization: means to improve access. Int J Gynaecol Obstet 2006;94: Gerrits T. Biomedical infertility care in low resource countries: barriers and access. Facts Views Vis Obgyn Monogr 2012;2: