Background: Due to rapid research progress in reproductive biology and reproductive clinical endocrinology, many human infertility treatments are close to potential breakthroughs and translational applications. We here review current barriers, where such breakthroughs will likely come from, what they will entail, and their potential clinical applications. Main text: The radical nature of change will primarily benefit older women, reduce fertility treatment costs and thereby expand access to treatment. A still widely overlooked prerequisite for implantation and normal pregnancy maintenance is timely development of maternal immunological tolerance toward an implanting paternal semi- allograft, if malfunctioning associated with implantation failure and pregnancy loss, while premature termination of tolerance appears associated with premature labor, pre-eclampsia/eclampsia and gestoses of pregnancy. Common denominators between pregnancy and invasive malignancies have again been attracting attention, suggesting that, like in malignant tumors, degrees of embryo aneuploidy may affect invasiveness and ability to “disarm” the immune system’s innate response against implanting embryos. Linking tolerance to implantation, we offer evidence that the so-called “implantation window” is likely immunological rather than hormonally defined. Conclusions: Because many here outlined treatment changes will disproportionally benefit older women, they will exert a pronounced effect on society, as increasing numbers of women at grandparental ages will become mothers. Keywords: Infertility, Gametes, Zygotes, Embryos, Cell lineage determination, Mosaicism, Maternal tolerance, Invasiveness, Implantation, Ovarian cycle Background paternity, and rather than disposed if genetically abnor- For reproductive biologists and reproductive clinical mal, embryos will be tested and, if abnormal, “repaired”, endocrinologist these are exciting times—possibly the utilizing newly developed genetic editing techniques most exciting times ever! At many different fronts, both . Successful in vitro maturation of primordial follicles disciplines appear on the verge of groundbreaking break- appears very close; preliminary models of oocyte-pro- throughs with remarkable potential impacts on fertility ducing artificial ovaries have already been reported [ 2]; treatments, prevention of genetic diseases but also on normal oocytes and spermatozoa have been produced other areas of medicine. in mice by reprogramming somatic cells into induced We foresee women having genetic offspring into ages pluripotent stem cells (iPSCs) and, then, into oocytes and beyond menopause, chemo- and radiotherapies no spermatozoa. Generations of healthy pups were the result longer being barriers to future genetic motherhood and . The same achievement in humans, is just a matter of time. Many of these developments will expand women’s reproductive lifespans. Considering likely cost sav *Correspondence: email@example.com The CHR, 21 East 69th Street, New York, NY 10021, USA ings, expanding affordability and access to fertility Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gleicher J Transl Med (2018) 16:149 Page 2 of 13 services—all highly desirable developments, changes will embryos and embryonic tissue was disallowed by Con- be disruptive in how fertility services will be provided. gress, while permitting research in the private sector . With older women already the most rapidly growing age Except for mandating from IVF centers annual reports to group having children , major societal adjustments the Center for Disease Control (CDC), the U.S. govern- must follow, affecting some of society’s most basic social ment mostly maintained a hands-off approach. and medical covenants: Schools will teach more children This changed in 2001 after attempts at cytoplasmic of older parents, and maternity services in hospitals will exchanges (cytoplasm from oocytes of young donors face more higher risk patients. Medical complications of was injected into oocytes of older women) were reported pregnancies will not only be more common but, at times, attempting to improve IVF outcomes, when the Food and also more severe . In other words, society will have to Drug Administration (FDA) declared regulatory author- adjust to a new generation of parents at what used to be ity over all embryo manipulations with potential effects grandparental ages. on the human germline. A briefing document distributed Disruptive changes to the fertility industry, there- to IVF on May 9, 2002 stated: We advised practitioners fore, must be anticipated [6, 7], likely even exceeding that FDA has jurisdiction over the use of human cells the changes brought about by the 1978 introduction of that have received transferred genetic materials by means in vitro fertilization (IVF) by Steptoe and Edwards . other than union of gamete nuclei. (https ://www.fda.gov/ IVF did radically revolutionize female as well as male fer-ohrms /docke ts/ac/02/ briefing/3855b1_01.pdf), thereby tility treatments, while with so-far almost seven million from this moment requiring Investigational New Drug IVF births worldwide, also changing the world. (IND) exemptions for all such studies. Since INDs are the Even scientists and physicians initially viewed IVF, process required for new drug approvals, this announce- however, with a degree of suspicion—some even consid- ment, instantly, made further research in this arena unaf- ered it ethically flawed. New potential accomplishments fordable for IVF centers. in human fertility treatments now face similar concerns: Since then, things got even worse: By, through a pro- “Organoids,” recently defined as, cells (that) have an vision of the Consolidated Appropriation Act, outright intrinsic ability to self-assemble and self-organize into prohibiting the FDA from even reviewing proposals complex functional tissues and organs , are increas- for INFs, Congress on December 18, 2015 reinserted ingly used as experimental in vitro substitutes for human itself even further . Existence of this moratorium is in vivo experiments. Because they mimic in vivo human extremely troublesome because it prevents in this area all organs and even human embryos (i.e., “embryoids”), they clinical research and practice in the U.S. have become controversial, leading to demands that, for Neither FDA nor Congress interposed themselves in ethical reasons, limits be imposed on their utilization. such fashion during the initial clinical evolution of IVF. Somewhat provocatively, some authors even renamed As long as IVF was considered “experimental,” super- organoids synthetic human entities with embryo-like fea- vision of clinical IVF programs by local Institutional tures (SHEEFs) . Interspecies-chimeric experimenta- Review Boards (IRBs) was considered adequate. Why tions have faced similar ethical concerns, despite their such a framework is no longer considered appropriate rather obvious importance for potential human organ- now is unclear. generations for transplantation purposes in other species This moratorium currently prevents clinical trials of [11, 12]. all cytoplasmic exchange procedures, whether for pre- Other potential innovations are also under intense vention of transmission of mitochondrial diseases from scrutiny , often held up by government regulations, mothers or for fertility purposes, and also applies to guidelines issued by professional bodies or quasi gov- Crispr-Cas9  and similar gene-editing procedures in ernment agencies  and/or national and international potential clinical applications to treat infertility. This was consensus agreements . Though ethical concerns are recently reaffirmed , when a New York IVF center especially warranted in experimentations with human performed spindle cell transfers utilizing donor cyto- reproduction, almost 40 years of IVF practice well dem- plasm  in preventing transmission of a mitochondrial onstrated the respect clinical and research communities disease from mother to offspring. By moving the embryo in reproductive biology and medicine have given to these transfer to Mexico, the investigators attempted to cir- concerns. cumvent the FDA’s regulatory authority. Considering that the embryo manipulation took place in a New York City, Government regulation the FDA, foreseeably , followed up with a “cease and IVF in the U.S. successfully evolved without government desist” letter . contributions. Already in 1973, years before birth of the Ultimately rewarded by a Nobel prize , worldwide first IVF offspring, future funding of research affecting success of IVF offers a good example, how reproductive Gleicher J Transl Med (2018) 16:149 Page 3 of 13 clinical and science communities can responsibly man- and cultured to blastocyst-stage . In 2013 Hayashi age ethically controversial issues with dignity and and Saitou reported generation of oocytes from mouse self-control without interference from government. ESCs and induced pluripotent stem cells (iPSCs) . Considering the overwhelming importance of human These experiments culminated in 2016 in a remarkable embryology research for stem cell sciences, regenerative report by Hikabe et al. in which the authors reconsti- medicine, immunology and oncology, at stake is no less tuted in vitro the entire cycle of the female germ line in than the country’s medical leadership in the world. The the mouse, in the process reconstituting oogenesis from U.S. will have to find ways to allow responsible research iPSCs, achieving fertilization and confirming down - to continue. stream multigenerational healthy progeny . In 2017 Hayashi et al. updated the methodology for reconstitut- Gametes ing from iPSCs mouse oogenesis by shortening the pro- Oocytes and spermatozoa at fertilization contribute (in cess to only approximately 5 weeks . Ishikura et al. in haploid format) their nuclear genomes (nDNA), while parallel reported derivation and propagation of spermat- oocytes also contribute the females’ mitochondrial ogonial stem cell activity in the mouse from iPSCs . genome (mDNA). This explains why mitochondrial dis - Similar experiments in humans, undoubtedly, are eases, caused by mutations of mDNA, are only passed on already underway. Once human primordial follicles can by mothers . be cultured to maturity in vitro, a small ovarian cortical Dogma still holds that women are born with all their biopsy at young age, yielding a few hundred primordial- follicles. Ovarian reserve (OR, describing quantity of stage follicles, could, thus, virtually guarantee lifelong remaining follicles/oocytes) constantly declines, start- fertility into advanced ages. iPSCs, reprogrammed from ing in utero . By age 51 (average time of menopause), peripheral skin cells, fibroblast, hair bulbs or other autol - follicles are in the low hundreds, and no longer respond ogous somatic cells, then directed toward autologous to gonadotropins. By producing fresh spermatozoa into oocytes and/or spermatozoa, would offer unlimited avail - very advance ages, males, in contrast, maintain fertility. ability of gametes even without need for ovarian or tes- In 2000, Xie and Spradling reported the existence of a ticular biopsies. germline stem cell (GSC) niche in ovaries of Drosophila Unlimited oocyte and sperm gluts will be highly dis- . Niches are regulatory microenvironments for stem ruptive . Developments most patients would greatly cells, created by stromal cells. Here, three somatic cells welcome, like women conceiving into much older ages, acted as a niche, able to replace GSCs, lost by normal or and obsolescence of ovarian stimulations via daily self- induced differentiation. injections of gonadotropins will make fertility treatments These observations encouraged the hunt for GSC more “patient-friendly” but will also greatly disrupt the niches in other species, including humans. Almost two pharma industry. Avoidance of the ovarian hyperstimula- decades later, GSCs are well characterized in non-mam- tion syndrome (OHSS), fortunately a rare complication of malian models, but their existence in mammals has ovarian hyperstimulation, can be viewed as improvement remained contested [26–28]. With stem cells in other in safety of treatments . human organs defined, lack of evidence for human GSCs Combining the use of iPSCs with the concept of repair- in ovaries is puzzling and inconsistent with current ing embryos with genetic defects (for more on this, see understanding of ovarian ontogeny. Their established next section), Hirota et al. demonstrated that reprogram- existence in non-mammalian animal models further sug- ming of stem cells from trisomic mice into iPSCs, tri- gests that they, simply, have not been properly identified somic cells returned to normal haploidy in a process the yet. authors named trisomy-based chromosome loss (TCL). This may have different causes: GSCs may be uncom - They then differentiated these now normal iPSCs into the mon and, therefore, difficult to isolate; currently known male germ line and functional sperm, producing chromo- markers may not identify them satisfactorily; or GSCs somally normal fertile offspring . Though performed may be identifiable by markers only while functionally in sterile XXY and XYY mice, the authors pointed out active, which they only rarely may be. Though existence that the technique was applicable to all trisomies, includ- of human GSCs, therefore, appears likely, they do not ing Down’s syndrome (Trisomy-21), the most frequent necessarily play a significant role in the reconstituting viable trisomy in humans, opening intriguing possibilities OR. for human treatments. In 2003, Hübner et al. generated in culture oogonia Their finding has also relevance for preimplantation from mouse embryonic stem cells (ESCs) that entered genetic screening (PGS), recently renamed preimplanta- meiosis, recruited adjacent cells to form follicle-like tion genetic testing for aneuploidy (PGT-A) [35, 36], and structures containing oocytes, which could be fertilized further addressed below. Gleicher J Transl Med (2018) 16:149 Page 4 of 13 Until very recently, any aneuploidy detected in embryos The zygote recently, however, attracted renewed atten - through PGS/PGT-A led to embryo disposal. In July of tion when Ma et al. reported improved success and accu- 2016, transfer guidelines were, however, radically revised, racy with Crispr-Cas9-editing in correcting a dominant now selectively permitting some transfers . mutation in human embryos causing hypertrophic cardi- The reasons are clinical observations following trans - omyopathy. They attributed their technical progress pri - fers of presumed aneuploid/mosaic embryos (discussed marily to concomitant intracytoplasmic sperm injection later) and stem cell data: For example, embryos with (ICSI) of oocytes and injections of Crispr-Cas9 . How abnormal PGS/PGT-A results can be source of euploid the defective gene was, however, corrected in the process stem cell lines [38, 39]. In reverse, normal human iPSCs was questioned, and their results have so-far not been also exhibit pervasive mosaic aneuploidies . Mouse duplicated. If confirmed the zygote, however, may assume experiments offered further evidence for the plasticity once again an important role in future gene-editing. of early-stage embryos by demonstrating chromosomal Similar to DNA editing with Crispr-Cas9, “base-edit- self-correction downstream from blastocyst-stage more ing,” may, at times, offer further advantages: Here, rather profoundly in the inner cell mass (ICM) than in trophec- than short strands of DNA, single base mutations of RNA toderm (TE) . Hirota et al. further defined this plas - are “corrected” , and message rather than germline ticity as a two-way street , and one is left wondering DNA is “edited.” Interventions, therefore, are temporary, about the purpose of ploidy determinations at blastocyst- and do not affect the germline by establishing perma - stage If embryos can self-correct further downstream nent multigenerational changes in DNA. “Base-editing,” [35, 36]. thus, offers potential new therapeutic opportunities for genetic editing, though with fewer short- as well as long- Zygotes term risks, including undesired permanent mosaicism at The single-cell organism formed at fertilization from offsite targets. Potential risks with Crispr-Cas9 and base- union of oocyte and sperm is a zygote. Its DNA is the editing, including off-target effects, however, do warrant combinations of both gametes’ genetic information, with caution before such techniques are applied upon human a haploid secondary oocyte and a haploid male gamete embryos in a clinical practice setting. unifying into a diploid cell. With the sperm entering the Through preimplantation genetic diagnosis (PGD), oocyte, the 2nd meiosis is completed. The result is a hap - current clinical practice allows diagnoses of hundreds loid maternal cell with half of its previous chromosomes of disease-causing single mutations in human embryos. (n = 23, nDNA), almost all of the oocytes original cyto- Affected embryos are then routinely discarded, a still plasm (including maternal mitochondria and, therefore controversial practice, by some considered reproductive mDNA), a haploid set of male chromosomes (n = 23, discrimination . Using above noted genetic editing nDNA) as male pronucleus, and an extruded second techniques, abnormal embryos should become “repair- polar body with another set of 23 of its chromosomes. able” and, therefore, transferrable. This would add to The nDNA of female and male pronuclei then replicates, size of available embryo pools for transfer and, therefore, temporarily creating a quadroploid (n = 23 × 4 chromo- improve pregnancy chances. Even current religious and somes) cell, thereby providing the substrate for fusion ethical opponents of IVF, should view avoidance of dis- of the two pronuclei ca. 30 h post-fertilization, and the posal of embryos positively. 1st mitotic division of the zygote into two diploid blasto- meres with 23 × 2 chromosomes each. Embryos Except during early days of IVF, when embryology Cleavage-stage embryo transfers on day-3 after fertiliza- laboratories were not yet able to culture embryos well tion (6–8 cell stage) replaced pronuclear (zygote) trans- in vitro, the zygote attracted limited attention in assisted fers once embryology laboratories improved embryo reproduction . As embryology improved, culture to culture conditions. By 1998, Gardner et al. however, cleavage-stage (day-3 after fertilization) became routine claimed blastocyst-stage transfers (on days-5/6 after fer- —until blastocyst-stage transfer was proposed by tilization) to improve clinical pregnancy rates and reduce Gardner et al. in the late 1990s . need for transfer of multiple embryos , thereby mini- A brief episode of secondary attention was awarded to mizing twin births. Though follow up studies did not the zygote when, initially through 1st polar body biopsy confirm claims of improved clinical pregnancy rates in and later through 1st and 2nd polar body biopsies com- unselected patients, later studies confirmed margin - bined , Verlinsky et al. proposed the concept of PGS. ally beneficial impacts on live birth rates, though only in Polar body biopsy was, however, manually too complex. good-prognosis patients . Universal blastocyst-stage Like embryo transfers before, PGS, therefore, quickly culture has, nevertheless, been gaining popularity ever migrated to cleavage and later to blastocyst-stage. since. Gleicher J Transl Med (2018) 16:149 Page 5 of 13 Best human embryos reach blastocyst-stage by day-5 Like human embryos, mouse embryos at cleavage stage after fertilization, and rarely by day-6. Sporadic preg- are characterized by seemingly “equivalent” blastomeres. nancies have even been reported from day-7 blastocysts. How these cells become either ICM or TE has remained Two recent human in vitro implantation studies [49, 50], controversial. To find the answer is important for a better demonstrated, without need for maternal contributions, understanding of early stages of human embryology—but normal human embryo development up to day 14 after has also clinical relevance for the increasingly popular fertilization. Human embryos, thus, appear self-regu- PGS/PGT-A procedure in association with clinical IVF. lated far beyond implantation. Experiments were only Here either 1–2 blastomeres (at cleavage-stage) or 5–7 terminated because international conventions currently TE cells (at blastocyst stage) are biopsied to determine still prohibit in vitro cultures of human embryos beyond whether embryos are euploid and, therefore, transferra- day-14. For how long human embryos can survive and ble or should be disposed. develop without maternal contributions is, therefore, still Under assumption of ICM and/or TE lineage-depend- unknown. ent biases, a TE-biopsy should more reliably reflect the Beyond current borders of viability (~ 22–24 weeks ges- ICM if assignments of initial blastomeres to cell lineages tational age), abilities to successfully maintain extremely are not biased but at random. Biased selection would, preterm infants are still poor. The par between already however, strongly suggest that TE biopsies cannot reli- demonstrated embryo culture abilities and potential neo- ably reflect ICMs. Cell-fate biases, indeed, appear initi - natal clinical viability, therefore, is only approximately ated as early as in the 2-cell stage and gaining pace at the 20–22 weeks. Using an ex vivo uterine environment, 4-cell stage. They derive from methylation of arginine 26 recent studies in very premature lambs demonstrated on histone 3 (H3R26), which determines length of bind- normal growth over a full week . “In vitro pregnancy” ing of important transcription factors to DNA. Longer (IVP), starting with IVF, and including long-term in vitro binding fosters expression of Sox21 (and other genes) and laboratory culture of embryos, followed by maintenance drives cells toward the embryonic (ICM) lineage, while until potential viability in an ex vivo system, therefore, cells with shorter exposure will develop toward the TE appears increasingly less utopian. Such an option appears (placental) lineage . also increasingly relevant, as women with absent uteri Most mouse studies reached the conclusion that such are increasingly exposed to risky and very costly uterine biases, indeed, exist [55, 58–60]. Niakan’s lab, however, transplants . recently again demonstrated that early embryo develop- We in this section, however, want to concentrate on ment distinctively varies in mice and men: By targeting two areas of research with considerable potential impor- and eliminating with Crispr-Cas9 in human zygotes the tance for the biological understanding of preimplanta- gene that encodes OCT4 (POU5F1), embryo develop- tion-stage embryos—cell lineage determinations and ment to blastocyst stage was compromised. In POU5F1- embryo mosaicism. null-cells, gene expression was then found downregulated for extra-embryonic TE genes (i.e., CDX2) as well as for regulators of the pluripotent epiblast (i.e., NANOG). Cell lineage determination In the mouse, elimination of pou5f1, however, did not The ability of one cell to give rise to all cell lineages (i.e., prevent blastocyst formation, though maintenance was mesoderm, endoderm, ectoderm and germ cells) defines impaired . The importance of OCT4 for human blas - pluripotency. In the human embryo, cells that have this tocyst-stage development was recently also confirmed by quality are a transient population, making up part of the Zernicka—Goetz’s laboratory . so-called epiblast (i.e., ICM) which, ultimately, forms the As embryos transit from pre-implantation to post- embryo/fetus/offspring. The early preimplantation-stage implantation stages, pluripotency within the epiblast embryo is made up of three distinctly different cell lin - declines (in the mouse, for a considerable time period eages—the embryonic epiblast, the extra-embryonic during gestation, it does not completely disappear), as primitive endoderm and the TE, which ultimately forms Fibroblast Growth Factor (FGF), Bone Morphogenic Pro- the placenta. tein (BMP) and other agents affect differentiation of epi - Though differences between human and mouse embryo blast cells into specialized and developmentally restricted development are coming into focus , the mouse is still fates. Rapidly evolving knowledge surrounding cell line- the principal subject of research [recently reviewed, 54, age determinations, will have crucially important con- 55]. The importance of studies in human embryos was, sequences for establishing fate-specific stem cell lines at however, recently reemphasized when OCT4 was dem- specific developmental stages [54, 55] and, with it, for onstrated to play distinctively different roles in mice and successful manipulations and reprogramming efforts of humans . cell populations, for establishing organoids and similar Gleicher J Transl Med (2018) 16:149 Page 6 of 13 cell-constructs for research [9, 62] and in treatments clinical pregnancy and live birth rates after transfer of of genetic diseases, cancers, as well as in regenerative embryos, by PGS/PGT-A reported to be aneuploid/ medicine. mosaic [68–72], offer, however, the single most convinc - ing evidence for such plasticity, and will, undoubtedly, Mosaicism lead to significant changes in embryology practice in IVF. How common mosaicism is in human preimplanta- tion embryos has remained controversial. Mosaicism is Tolerance defined as presence of more than once chromosomal cell Implanting embryos are paternal semi-allografts. The lineage in a tissue, organ or embryo/individual. Because maternal immune system, therefore, should reject them; of a process called “microchimerism,” most, if not all yet, in normal pregnancies competent maternal immune humans, are chimeras, as during intrauterine life mosaic systems do not reject implanting embryos. They, there - clones are routinely transmitted from mothers to off - fore, must reprogram themselves from rejection to tol- spring and vice versa . erance. Timely development of maternal tolerance, Because of its alleged ability to improve outcomes, uti- therefore, must be viewed as an absolute prerequisite for lization of PGS/PGT-A in association with IVF has been successful implantation and pregnancy maintenance. increasing. Three consecutive generations of the proce - Induction of maternal tolerance differs from induction dure so-far have, however, been unable to demonstrate of tolerance in solid organ recipients, where allogeneic promised outcome benefits. We [35, 36] and others  antigen load is consistent, while in pregnancy it grows have argued that significant underestimates of TE-mosa - exponentially with advancing gestation. Also, organ icism have been a principal reason, resulting in large transplantation requires permanent tolerance, while numbers of false-positive diagnoses  and wasteful in pregnancy tolerance is only temporary (on average disposal of transferrable embryos. Especially in poorer 40 weeks from last menstrual period). prognosis patients with only small embryo numbers, Whether premature or at term, labor is increasingly erroneous disposal of healthy embryos will actually nega- considered caused by termination of this temporary tively affect outcomes. tolerance . Appropriate maternal tolerance levels, TE-mosaicism in blastocyst-stage embryos may, therefore, appear essential from implantation until labor. indeed, be almost universal, and fulfill important physi - What produces the remarkable biological characteristics ological functions. In malignant tumors, degrees of ane- of maternal tolerance toward the paternal semi-allograft uploidy correlate with invasiveness and obfuscation of is, however, still largely unknown and is currently actively patients’ immune responses to tumors . Aneuploid/ pursued in a number of research laboratories around the mosaic cell clones in TE, therefore, may be supportive of world. the invasive implantation process . Paradoxically, research efforts have primarily concen - Additional observations have contributed to increas- trated on local immune responses within the complex ing skepticism about the efficacy of PGS/PGT-A. As cur - micro-environments of implantation sites . Yet, since rently practiced, PGS/PGT-A involves TE-biopsies at implantation sites never demonstrate evidence of allo- blastocyst-stage. Since TE represents the placental cell geneic immune responses (even if implantation occurs lineage, the procedure assumes that biopsies of the pla- extra-uterine), adequate tolerance levels must exist even cental precursor structure offer reliable chromosomal before implantation occurs. Though some of the pre - information about epiblast (ICM), the fetal precursor sumed steps in tolerance induction we here describe representative of all three future germ layers and germ are still unproven, basic knowledge of how the immune cells. system functions, supports a cascade of events, as here Discrepancies between TE and ICM in human embryos described. have, however, been reported . In TE, but espe- What happens (in the micro- environment of implan- cially profoundly in the ICM, embryos also demonstrate tation sites) immediately after implantation must, there- remarkable plasticity capable of eliminating aneuploid fore, already represent a second stage in development of cell clones downstream from blastocyst stages . maternal tolerance. In absence of a yet undescribed first Though so-far directly only demonstrated in mice, evi - step in tolerance induction, implantation would either dence for similar human plasticity can be deducted: never happen or evidence of an allogeneic immune Embryos reported as aneuploid/mosaic by PGS/PGT-A response must be visible. Women who lack this first stage have given rise to normal euploid stem cell lines [38, 39], tolerance level, likely, indeed, either do not implant (i.e., while, at the opposite extreme, stem cells from trisomic suffer from implantation failure) or miscarry so early that mice returned to a haploid state when reprogrammed pregnancy is either not recognized or cannot be histolog- into iPSCs, . Surprisingly excellent implantation, ically assessed (i.e., chemical pregnancies). Gleicher J Transl Med (2018) 16:149 Page 7 of 13 For decades investigators have hypothesized that period, or in previously mentioned in vitro implantation abnormal maternal immune function may be responsible models [49, 50] may, therefore, be revealing. for implantation failures  and pregnancy losses . Timely induction of tolerance pathways, then allows Presumed abnormal immune responses were, however, implanting embryos to invade the endometrium (or, in considered to be autoimmune—thus mistakenly leading cases of extra-uterine pregnancies, other tissues). Exist- to treatment of presumed autoimmune- rather than allo- ence of such pathways can be deduced from another immune responses , while failing to recognize that experiment of nature: Immune responses to selected hel- insufficient initial tolerance development was the real ini - minths can positively or negatively affect female fecun - tial culprit causing maternal allogeneic responses. dity . Like the fetal semi-allograft, helminths are In microenvironments around implantation sites, local parasites, their survival depending on adequate toler- immune responses also play important roles as part of a ance levels of the host. Some helminths appear to induce cascade of sequential tolerance-inducing layers that offer similar tolerance pathways to those induced by embryos. timely tolerance and the necessary redundancies that Women infected with those parasites, therefore, demon- characterize all essential biological processes. Systemic strated improved fecundity. tolerance pathways, likely induced during the 48 h imme- Based on these observations, we predict the discov- diately preceding implantation are, however, probably ery of treatments that will induce tolerance-inducing most essential for early implantation. Extra-uterine preg- pathways, which will decrease implantation failures and nancies, which in extreme cases can go to term , are miscarriage rates and significantly reduce the incidence the most convincing “natural experiment” in support of of premature labor especially in women with inflamma - this assumption since they demonstrate convincingly that tion and autoimmune diseases, where prematurity is an adequate tolerance levels needed for implantation evolve almost universal phenomenon . even in absence of uteri. Once embryos invade the maternal host (whether via Invasiveness the endometrium or at extra-uterine sites), systemic tol- Like malignant tumors, implanting embryos possess erance pathways are then, likely, further augmented by invasive properties, and in both clinical circumstances local immune responses within the invasive microenvi- a seemingly normally functioning immune system is ronments surrounding the implanting embryos. Facing circumvented. Except in malignant gestational tumors, a rapidly growing fetus and placenta and the challenges embryos, however, quickly lose their invasiveness, while of a logarithmically growing antigenic mass, tolerance is, malignancies not only retain but, often, accelerate their likely, further augmented by maternal and fetal micro- invasive capabilities over time. Discovering the turn- chimerism . Starzl was the first to demonstrate that off switch for time-limited invasiveness in implanting mutual microchimerism in donor organ and organ recip- embryos, therefore, could have major relevance for can- ient was important for successful allogeneic solid organ cer therapeutics. transplantation . It, likely, is equally important in Analogies between cancer and embryo invasiveness establishing temporary tolerance in human pregnancy. go further: Aneuploidy is a hallmark of invading tumors, A desensitizing effect of semen on the maternal found in 90% of solid tumors , a percentage quite sim- immune system has been suspected for decades since ilar to what now is considered mosaic aneuploidy in TE prior exposure to semen appears inversely associated at blastocyst-stage. In cancer, the quantity of aneuploidy with preeclampsia/eclampsia risk, [78, 79]. Since preg- in a tumor correlated to its invasiveness. Whole-arm or nancy chances in virgins are not reduced, semen expo- whole-chromosome somatic copy number alterations sure, alone can, however, not be presumed to induce (SCNAs) affect ca. 25% of the genome of a cancer cell, sufficient tolerance. Like other essential processes in while focal SCNAs affect ca. 10% . human reproduction, development of adequate toler- A tumor’s degrees of aneuploidy also correlate with ance, likely, depends on multiple inductive processes, its abilities to disarm a host’s immune responses against which not only work in sequence but also serve as bio- the invading tumors. The greater the aneuploidy, the less logical back-up systems, in cases one fails. immune systems resist invading tumors . Highest Embryos spend approximately 48 h “floating” within levels of aneuploidy in a malignancy (> 70th percentile of the microenvironment of the uterine cavity before SCNA score) were associated with reduced T cell num- implanting. This time period must have functional bers but increased immune suppressive macrophages, importance and can be assumed to allow communica- thus controlling the tumor’s micro-environment and tions between embryos and maternal immune systems. immune components independently of affecting selec - Investigations of secretory products during this time tion of the T cell repertoire by neoantigen epitopes. Gleicher J Transl Med (2018) 16:149 Page 8 of 13 T cells attack tumors in various ways. One suppres- that human embryos apparently developed normally sive mechanism was recently identified in drug-resist - up to post-fertilization day-14 in in vitro culture lack- ant malignant gestational trophoblastic disease (where ing endometrium and/or maternal contributions [49, invasiveness of fetal tissue apparently does not shut off ) 50]. That implantation depends on specific qualities of : As in other solid tumors , Pembrolizumab, a maternal endometrium, therefore, appear increasingly monoclonal antibody that blocks the tumor-expressed unlikely. More likely, once embryos enter the endome- programmed cell death ligand 1 (PD-L1) signaling to the trial cavity, it is them who affect the endometrium. T cell inhibitory receptor (programmed death protein In a mouse model, microenvironments in both uter- 1[PD-1]) pathway, was found clinically effective in inhib - ine horns developed very differently depending on iting tumor growth. whether cleavage-stage or blastocyst-stage embryos In mice, PD-L1 expression has been demonstrated were transferred into a uterine horn , suggesting to maintain gestational tolerance, while loss of PD-L1 that, likely “educated” by signals from “entrance-seek- signaling resulted in fetal rejection . Since PD-L1 ing” embryos, endometrial plasticity adjusts to devel- is strongly expressed in trophoblastic malignancies, opmental stages of embryos. Communications between it is suspected to be involved in certain solid tumors’ embryos and endometrium during those 48 h of “floata - immune-evasive properties . Like tumor cells, blas- tion” in the endometrial cavity before implantation tocysts of early stage human embryos in general dem- are also suggested by in vitro studies, suggesting an onstrate excessive expression of gene products that favor innate ability of interstitial endometrial cells to “sense” cell progression, while not demonstrating expression of embryo quality, and favor good over bad [92, 93]. cell cycle check point genes . Combining immunology, invasiveness and here noted Further strengthening the analogy, microenvironments endometrial plasticity in response to embryonic guid- of implanting embryos demonstrate on multiple levels ance into a new all-encompassing implantation hypoth- remarkable similarities to microenvironments of malig- esis, it appears that the endometrium, in fulfilling an nant tumors. Exactly as seen in malignancies, TE ane- innate immunobiological function of protecting women uploidy may, thus, not only relate to invasiveness of early from bacteria, parasites, cancer cells and other poten- stage embryos (i.e., implantation capacity) but also to tially harmful invaders, is always “hostile” and, there- how embryos may affect local immune responses within fore, also “hostile” to implantation. This immunological microenvironments of implantation sites. Paradoxically, hostility is only overcome, once invaders, like parasites this would invert the current PGS/PGT-A hypothesis,  or semi-allogeneic embryos establish adequate sys- as more aneuploid TEs might then denote better rather temic immunological tolerance, and uteri (or in cases than poorer implantation chances. of extra-uterine pregnancies other organs), therefore, Using whole transcriptome microarrays and enrich- become permissible to implantation. ment analyses with GO gene sets, a recent mouse study, Implantation, thus, appears driven mostly by biologi- comparing global fetal tissues and tumor microenviron- cally desirable embryos, with implantation sites being ments, supported these conclusions: Central pathways reactive, rather than proactive. Considering timing of toward tolerance induction in both clinical circum- entry of embryos into the endometrial cavity, adequate stances were remarkable similar in antigen-presentation, tolerance development usually, still, falls on cycle days lymphocyte activation and T-regulator cell (Treg) activa- 20–24 (6–10 days after ovulation), and “synchrony,” tion . indeed, still appears to fall within 3.0 ± 1.5 days; those are, however, secondary effects and, because of endo - metrial plasticity, have limited clinical relevance. Implantation Primarily, implantation must be viewed as an immuno- Improved understanding of immunology and invasive- logically- rather than endocrinologically-determined ness of human embryo implantation also raises questions process. about the so-called (hormonally-determined) implan- An immunological definition of implantation clini - tation window, defined by cycle days 20–24 (6–10 days cally, however, demands new diagnostic capabilities after ovulation) [88, 89], a hypothesis requiring syn- of defining what represents adequate tolerance and, chronization between endometrium and embryo devel- therapeutically, how tolerance pathways of pregnancy opment, with asynchrony of greater than 3.0 ± 1.5 days can be boosted when inappropriately low, and leading resulting in infertility . to implantation failure, miscarriages and/or late preg- To a degree, the high prevalence of extra-uterine nancy complications, like premature labor . pregnancies in humans, however, contradicts this hypothesis. So is also the previously noted observation Gleicher J Transl Med (2018) 16:149 Page 9 of 13 The ovarian cycle events . Further improvements, therefore, must come Since introduction of gonadotropin therapy approxi- from interventions into earlier stages of follicle matura- mately six decades ago, female infertility treatments tion, including manipulations of follicle recruitments out almost exclusively only concentrated on the last 2 weeks of the primordial follicle pools of resting follicles . of follicle maturation, the so-called gonadotropin- Such efforts have been under way for some time, as the dependent stages of folliculogenesis. Treatment was mechanisms underlying follicle recruitment are increas- oriented toward the monthly menstrual cycle and, there- ingly well understood, with the mTor pathway appar- fore, treatment cycles were defined by the approximately ently playing an important role . Concomitantly, monthly interval between two menstrual periods. the importance of anti-Müllerian hormone (AMH) in Once recruited out of resting stage (primordial folli- restraining recruitment has been better elucidated . cles), a follicle’s journey of maturation toward gonadotro- The utility of a “monthly” treatment cycle, therefore, pin dependency takes at minimum two, and possibly as becomes questionable, instead suggesting a new defi - long as 4 months. Pharmacological interventions in the nition of what constitutes a treatment cycle as the time gonadotropin-dependent phase, therefore, occur rela- period it takes for follicles to mature between initial tively late, when follicle and oocyte quality (and quantity) recruitment up to ovulation and/or retrieval in an IVF to a large degree have already been determined by earlier cycle (Fig. 1). I— RESTING POOL—II--------------------------------------------- FSH SENSIIVITY --------------------------------------------I I --FSH DEPENDENCY---I I--SMALL GROWING FOLLICLE POOL--I I—LARGE GROWING FOLLICLE POOL--I Fig. 1 Folliculogenesis. The process of folliculogenesis, the time a single recruited follicle takes to reach ovulation (or retrieval in an IVF cycle, assuming it does not undergo atresia and apoptosis before) extends over multiple menstrual cycles. From a follicle’s vantage point, a real treatments cycle, therefore, involves multiple menstrual cycles. With therapeutic interventions, like androgen and HGH supplementations, moving into earlier stages of follicle maturation that the last 2 weeks of FSH-dependency, what is considered a treatment cycle, therefore, requires reevaluation Gleicher J Transl Med (2018) 16:149 Page 10 of 13 Some efforts along these lines have been underway for protocols, allowing stimulated cycle starts in either follic- over a decade. For example, androgen supplementation in ular or luteal phases, and even back to back double-stim- women with low ovarian reserve increased in popularity ulations in follicular and luteal phases [100, 101]. Both of once importance of adequate testosterone levels at small these practice changes are based on the recognition that growing follicle stages became apparent in enhancing follicle maturation is a constant process; some follicles, FSH-sensitivity of granulosa cells . This first example therefore, reach gonadotropin dependency every day. of earlier intervention into folliculogenesis also demon- Going forward, such an updated definition of the ovar - strates the importance of ovarian microenvironments for ian cycle should also enhance ongoing efforts to learn to follicle maturation and raised interesting questions about control this cycle in vitro. currently still widely held believes as to what constitutes ovarian aging. As with advancing age female fecundity declines, ova- Conclusions ries are believed to age because of constant loss of finite We here outlined some of the major developments we follicle/egg numbers and declining (mostly chromo- predict to enter clinical practice within the foreseeable somal) quality of oocytes. Androgen supplementation future. Some will be truly groundbreaking—revolution- reports, however, suggest that declining oocyte quan- ary, while others may appear more evolutionary. tity and quality may not be consequence of only oocyte Reviewing the modern history of fertility treatments, aging. That raising androgen levels in hypo-androgenic only three events can be characterized as truly ground- women improves ovarian function, pregnancy and live breaking: The introduction of gonadotropin therapy birth chances, instead suggests that reconstitution of by Gemzell  and Lunenfeld , as already noted aging ovarian microenvironments, in which follicles IVF  and, likely, the addition of intracytoplasmic mature, may at least partially reverse ovarian aging. In sperm injection (ICSI) to IVF by Palermo et al.  in other words, at least part of ovarian aging is consequence 1992, which offered genetic paternity to almost all male of aging of the ovarian micro-environment rather than of infertility patients. These three new treatments were oocytes. groundbreaking because each of them allowed large new Difference between these two ovarian aging hypotheses groups of previously infertile, and often sterile, couples to are of great theoretical and practical importance because become parents. No other fertility treatments can make any physical damage from presumed aging of oocytes this claim. within primordial follicles must be considered irrevers- Advancing female age, proportionally and progres- ible; as androgen supplementation demonstrated, recon- sively, has assumed ever larger swats of the fertility land- stitution of ovarian microenvironments with due to age scape. In developed countries, infertility in older women insufficient components, is, however, possible. This new has in recent years become the quintessential infertil- hypothesis of ovarian aging developed at New York’s ity problem, as steadily improving IVF outcomes allow Center for Human Reproduction, therefore, for the first younger patients relatively quick conceptions, while older time suggests that in some women quality of oocytes can women often linger on in the system. be improved, while the traditional hypothesis of ovarian Older women will, however, be the primary beneficiar - aging offers no such option. ies of here discussed impending outcome improvements Therapeutic interventions into earlier stages of fol - from translational clinical applications of recent research liculogenesis, however, also suggest a new definition for developments. As also noted, with ever older women what constitutes an ovarian treatment cycle as the time conceiving, major societal changes will follow, for which between follicle recruitment and ovulation and/or oocyte medicine and society must appropriately prepare. Here retrieval in IVF cycles and, therefore, includes multiple described likely new developments now appear closer menstrual cycles (Fig. 1). than even only a few years ago. Potential impacts on soci- Clinical practice increasingly points toward acceptance ety, therefore, likely will occur sooner and at more rapid of this concept. Again, androgens are a good example: pace than previously anticipated. Since androgen supplementation primarily benefits small Because the early embryo contains all the genetic infor- growing follicles , those follicles still require weeks– mation of a lifetime of health and disease, discoveries in months to reach gonadotropin-dependency and ovula- reproductive biology often impact many other areas of tion. Androgen supplementation, therefore, must be human medicine. Developments in reproductive biol- initiated weeks–months before affected follicles/oocytes ogy, therefore, will not only benefit human reproduction become available to gonadotropin stimulation and should but all of medicine. It is for that reason that continu- be carried through till cycle completion . A second ous uninhibited ethical research in reproductive biol- example are recently reported new ovarian stimulation ogy and reproductive clinical medicine is of such crucial Gleicher J Transl Med (2018) 16:149 Page 11 of 13 microporous scaffolds restores ovarian function in sterilized mice. Nat importance, if the U.S. is to maintain its leadership posi- Commun. 2017;16(8):15261. tion in medical sciences. 3. Hikabe O, Hamazaki N, Nagamatsu G, Obata Y, Hirao Y, Hamada N, Shi- mamoto S, Imamura T, Nakashima K, Saitou M, Hayashi K. Reconstitut- ing in vitro of the entire cycle of the mouse female germ line. Nature. Abbreviations 2016;539(7628):299–303. BMP: bone morphogenic protein; CDC: Center for Disease Control; ESC: 4. Gleicher N, Kushnir VA, Weghofer A, Barad DH. The “graying” of infertility embryonic stem cell; FDA: Food and Drug Administration; GSC: germline services: an impending revolution nobody is ready for. Reprod Biol stem cell; iPSC: induced pluripotent stem cell; IVF: in vitro fertilization; mDNA: Endocrinol. 2014;9(12):63. mitochondrial DNA; nDNA: nuclear DNA; FGF: fibroblast growth factor; ICM: 5. 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Addressing the ethical issues School, 1090 Vienna, Austria. raised by synthetic human entities with embryo-like features. Elife 2017;6. Pii: e20674; Erratum in Elife2017;6. Pii: e27642. Acknowledgements 11. Wu J, Platero Luengo A, Gil MA, Suzuki K, Cuello C, Morales Valencia M, Nor applicable. Parrilla I, Martinez CA, Nohalez A, Roca J, Martinez EA, Izpisua Belmonte JC. Generation of human organs in pigs via interspecies blastocyst Competing interests complementation. Reprod Domest Anim. 2016;51(Suppl 2):18–24. N.G. is a co-inventor on a number of pending and already awarded U.S. 12. Wu J, Platero-Luengo A, Sakurai M, Sugawara A, Gil MA, Yamauchi T, patents claiming therapeutic benefits from androgen supplementation in Suzuki K, Bogliotti YS, Cuello C, Morales Valencia M, Okumura D, Luo J, women with low functional ovarian reserve (LFOR) and relating to the FMR1 Vilariño M, Parrilla I, Soto DA, Martinez CA, Hishida T, Sánchez-Bautista gene in a diagnostic function in female fertility. 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Journal of Translational Medicine
– Springer Journals
Published: Jun 4, 2018