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Accurate chromosome segregation during meiosis is crucial for reproduction. Human and porcine oocytes transiently cluster their chromosomes before the onset of spindle assembly and subsequent chromosome segregation. The mechanism and function of chromosome clustering are unknown. Here we show that chromosome clustering is required to prevent chromosome losses in the long gap phase between nuclear envelope breakdown and the onset of spindle assembly, and to promote the rapid capture of all chromosomes by the acentrosomal spindle. The initial phase of chromosome clustering is driven by a dynamic network of Formin-2- and Spire-nucleated actin cables. The actin cables form in the disassembling nucleus and migrate towards the nuclear centre, moving the chromosomes centripetally by interacting with their arms and kinetochores as they migrate. A cage of stable microtubule loops drives the late stages of chromosome clustering. Together, our data establish a crucial role for chromosome clustering in accurate progression through meiosis.

Recent advances in single cell omics have been transformative in the characterisation of challenging to study biological contexts, including when the source material is precious, such as the early human embryo. Single cell datasets are technically challenging to infer transcription factor-gene regulatory interactions, due to low read-depth leading to zero inflated data. Here we have systematically assessed the application of four different machine learning linear or non-linear gene regulatory network prediction strategies to single cell simulated and human embryo transcriptome datasets. We have also compared how the method of gene expression normalisation impacts on regulatory network predictions. Integrating chromatin accessibility together with transcript expression datasets improved the reproducibility of the predicted gene regulatory networks. We found that the application of a non-linear network prediction method based on mutual information (MI) to single cell transcriptome datasets refined with chromatin accessibility (CA) (called MICA), exhibited higher reproducibility compared to the alternative network interference methods tested. Moreover, MICA was used to make predictions about GRNs in the preimplantation human embryo, which were supported by previous findings in other developmental and stem cell contexts. Based on the gene regulatory networks predicted by MICA, we discovered co-localisation of the AP-1 transcription factor subunit proto-oncogene JUND and the TFAP2C transcription factor AP-2γ in human preimplantation embryos. Overall, our comparative analysis of gene regulatory network prediction methods defines a pipeline that can be applied to single-cell multi-omics datasets to infer interactions between transcription factor expression and target gene regulation, which can then be functionally tested.

Full-grown oocytes are transcriptionally silent and must stably maintain the messenger RNAs (mRNAs) needed for oocyte meiotic maturation and early embryonic development. However, where and how mammalian oocytes store maternal mRNAs is unclear. Here, we report that mammalian oocytes accumulate mRNAs in a mitochondria-associated ribonucleoprotein domain (MARDO). MARDO assembly around mitochondria was promoted by the RNA-binding protein ZAR1 and directed by an increase in mitochondrial membrane potential during oocyte growth. MARDO foci coalesced into hydrogel-like matrices that clustered mitochondria. Maternal mRNAs stored in the MARDO were translationally repressed. Loss of ZAR1 disrupted the MARDO, dispersed mitochondria, and caused a premature loss of MARDO-localized mRNAs. Thus, a mitochondria-associated membraneless compartment controls mitochondrial distribution and regulates maternal mRNA storage, translation, and decay to ensure fertility in mammals.

Our understanding of the molecular events driving cell specification in early mammalian development relies mainly on mouse studies, and it remains unclear whether these mechanisms are conserved across mammals, including humans. We have recently shown that the establishment of cell polarity via aPKC is a conserved event in the initiation of the trophectoderm (TE) placental program in mouse, cow, and human embryos. However, the molecular mechanisms transducing cell polarity into cell fate in cow and human embryos is unknown. Here, we have examined the evolutionary conservation of the molecular cascade downstream of aPKC in four different mammalian species: mouse, rat, cow, and human. Surprisingly, by morphokinetic and immunofluorescence analyses, we observe that rat embryos more closely recapitulate human and cow developmental dynamics, in comparison to the mouse. Nevertheless, in all four species, inhibition of the Hippo pathway by targeting LATS kinases is sufficient to drive ectopic TE initiation and downregulation of SOX2, a marker of the inner cell mass. Our comparative embryology approach uncovered intriguing differences as well as similarities in a fundamental developmental process among mammals, reinforcing the importance of cross-species investigations.

Human oocytes are prone to assembling meiotic spindles with unstable poles, which can favor aneuploidy in human eggs. The underlying causes of spindle instability are unknown. We found that NUMA (nuclear mitotic apparatus protein)–mediated clustering of microtubule minus ends focused the spindle poles in human, bovine, and porcine oocytes and in mouse oocytes depleted of acentriolar microtubule-organizing centers (aMTOCs). However, unlike human oocytes, bovine, porcine, and aMTOC-free mouse oocytes have stable spindles. We identified the molecular motor KIFC1 (kinesin superfamily protein C1) as a spindle-stabilizing protein that is deficient in human oocytes. Depletion of KIFC1 recapitulated spindle instability in bovine and aMTOC-free mouse oocytes, and the introduction of exogenous KIFC1 rescued spindle instability in human oocytes. Thus, the deficiency of KIFC1 contributes to spindle instability in human oocytes.

Current knowledge of the transcriptional regulation of human pluripotency is incomplete, with lack of interspecies conservation observed. Single-cell transcriptomics analysis of human embryos previously enabled us to identify transcription factors, including the zinc-finger protein KLF17, that are enriched in the human epiblast and naïve human embryonic stem cells (hESCs). Here, we show that KLF17 is expressed coincident with the known pluripotency-associated factors NANOG and SOX2 across human blastocyst development. We investigate the function of KLF17 using primed and naïve hESCs for gain- and loss-of-function analyses. We find that ectopic expression of KLF17 in primed hESCs is sufficient to induce a naïve-like transcriptome and that KLF17 can drive transgene-mediated resetting to naïve pluripotency. This implies a role for KLF17 in establishing naïve pluripotency. However, CRISPR-Cas9-mediated knockout studies reveal that KLF17 is not required for naïve pluripotency acquisition in vitro. Transcriptome analysis of naïve hESCs identifies subtle effects on metabolism and signalling pathways following KLF17 loss of function, and possible redundancy with other KLF paralogues. Overall, we show that KLF17 is sufficient, but not necessary, for naïve pluripotency under the given in vitro conditions., Summary: Given that KLF17 was shown to be sufficient, but not necessary, to establish naïve pluripotent hESCs, KLF17 might function as a peripheral regulator of human pluripotent stem cells.

During the first week of development, human embryos form a blastocyst comprised of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles and directed differentiation potential similar to existing human TSCs. Systematic omission of each or combinations of factors, revealed the critical importance of GATA2 and GATA3 for successful iTSC reprogramming. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases.Summary statementTranscriptional analysis of human blastocysts reveals transcription factors sufficient to derive induced trophoblast stem cells from primed human embryonic stem cells.

SummaryAssisted reproductive technology is today considered a safe and reliable medical intervention, with healthy live births a reality for many IVF and ICSI treatment cycles. However, there are increasing numbers of published reports describing epigenetic/imprinting anomalies in children born as a result of these procedures. These anomalies have been attributed to methylation errors in embryo chromatin remodelling during in vitro culture. Here we re-visit three concepts: (1) the so-called ‘in vitro toxicity’ of ‘essential amino acids’ before the maternal to zygotic transition period; (2) the effect of hyperstimulation (controlled ovarian hyperstimulation) on homocysteine in the oocyte environment and the effect on methylation in the absence of essential amino acids; and (3) the fact/postulate that during the early stages of development the embryo undergoes a ‘global’ demethylation. Methylation processes require efficient protection against oxidative stress, which jeopardizes the correct acquisition of methylation marks as well as subsequent methylation maintenance. The universal precursor of methylation [by S-adenosyl methionine (SAM)], methionine, ‘an essential amino acid’, should be present in the culture. Polyamines, regulators of methylation, require SAM and arginine for their syntheses. Cystine, another ‘semi-essential amino acid’, is the precursor of the universal protective antioxidant molecule: glutathione. It protects methylation marks against some undue DNA demethylation processes through ten-eleven translocation (TET), after formation of hydroxymethyl cytosine. Early embryos are unable to convert homocysteine to cysteine as the cystathionine β-synthase pathway is not active. In this way, cysteine is a ‘real essential amino acid’. Most IVF culture medium do not maintain methylation/epigenetic processes, even in mouse assays. Essential amino acids should be present in human IVF medium to maintain adequate epigenetic marking in preimplantation embryos. Furthermore, morphological and morphometric data need to be re-evaluated, taking into account the basic biochemical processes involved in early life.

Following implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development., Single cell analysis of early human embryos identifies key changes in pluripotency, the requirement of FGF signalling for embryo survival, and defines a putative anterior-like region of hypoblast cells, providing insights into how early human development is regulated.

Medically assisted reproduction, now considered a routine, successful treatment for infertility worldwide, has produced at least 8 million live births. However, a growing body of evidence is pointing toward an increased incidence of epigenetic/imprinting disorders in the offspring, raising concern that the techniques involved may have an impact on crucial stages of early embryo and fetal development highly vulnerable to epigenetic influence. In this paper, the key role of methylation processes in epigenesis, namely the essential biochemical/metabolic pathways involving folates and one-carbon cycles necessary for correct DNA/histone methylation, is discussed. Furthermore, potential contributors to epigenetics dysregulation during the three phases of assisted reproduction: preparation for and controlled ovarian hyperstimulation (COH); methylation processes during the preimplantation embryo culture stages; the effects of unmetabolized folic acid (UMFA) during embryogenesis on imprinting methyl “tags”, are described. Advances in technology have opened a window into developmental processes that were previously inaccessible to research: it is now clear that ART procedures have the potential to influence DNA methylation in embryonic and fetal life, with an impact on health and disease risk in future generations. Critical re-evaluation of protocols and procedures is now an urgent priority, with a focus on interventions targeted toward improving ART procedures, with special attention to in vitro culture protocols and the effects of excessive folic acid intake.

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac1. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human2–5 and cow6. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical–basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos. Comparative analysis of human, cow and mouse embryos shows that a mechanism involving atypical protein kinase C initiates the trophectoderm program during the morula stage in these three species.

The vast majority of human embryos are aneuploid. Aneuploidy frequently arises during the early mitotic divisions of the embryo, but the origin of this remains elusive. Using bovine embryos as a model for human embryos, we identify an error-prone mechanism of parental genome unification which often results in aneuploidy. Surprisingly, genome unification initiates hours before breakdown of the two pronuclei that encapsulate the parental genomes. While still within intact pronuclei, the parental genomes polarize towards each other, in a process driven by centrosomes, dynein, and microtubules. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in direct proximity to each other. Parental genome clustering often fails however, leading to massive chromosome segregation errors, incompatible with healthy embryo development. Nucleoli, which associate with chromatin, also cluster at the pronuclear interface in human zygotes. Defects in nucleolar clustering correlate with failure in human embryo development, suggesting a conserved mechanism.

Life expectancy has increased since World War II, and this may be attributed to several aspects of modern lifestyles. However, now we are faced with a downturn, which seems to be the result of environmental issues. This paradigm is paralleled with reduced human fertility, decreased sperm quality, increased premature ovarian failure, and diminished ovarian reserve syndromes. Endocrine disruptor chemicals and other toxic chemicals, herbicides, pesticides, plasticizers, to mention a few, are a rising concern in today's environment. Some of these are commonly used in the domestic setting: cleaning material and cosmetics and they have a known impact on epigenesis and imprinting via perturbation of methylation processes. Pollution from polyaromatic hydrocarbons, particulate matter

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

DNA methylation is a biochemical process that modifies gene expression without changing the underlying DNA sequence, and this represents the molecular basis for imprinting and epigenetics. Recent reports have revealed alterations in DNA methylation profiles in the placenta of babies born from assisted reproductive technologies (ART). This supports several previous observations that suggested an increase in the prevalence of imprinting diseases following ART treatment, and also fits our observations regarding the metabolism and requirements of early human embryos. Human embryo culture media (HECM) are currently formulated according to requirements based on the mouse embryo model, and in fact need to pass the Mouse Embryo Assay test in order to be accepted by the relevant authorities, despite the fact that physiological (especially the time necessary to reach genomic activation) and biochemical requirements of mouse and human embryos are quite different. This commentary aims to explain some of the discrepancies, and emphasize why human embryo metabolism tells us that the composition of HECM, as well as the role of the MEA as a unique model, should be re-evaluated.

Understanding fertility in young and old Fertility in humans follows a U-curve, with low rates in both teenagers and women of advancing maternal age (mid-30s and above). Gruhn et al. found that this distinct shape originates from chromosomal errors in human eggs, which result in genomic imbalance and pregnancy loss. The error types and chromosomes affected in the young and advanced age groups were different, suggesting that two distinct chromosome-based mechanisms balance risk associated with pregnancy and evolutionary fitness as women enter and exit their reproductive life span. The authors show that chromosome structure erodes only with advancing age, acting as a “molecular clock” for reproductive senescence. Science , this issue p. 1466

Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche., The signals regulating the establishment and maintenance of the pluripotent epiblast in human embryos are unclear. Here, the authors use a bioinformatics approach to identify the role of IGF1 in human embryo development, and from this, propose a culture medium with IGF1 together with Activin to sustain hESCs in the absence of FGF.

Summary Most human embryos are aneuploid. Aneuploidy frequently arises during the early mitotic divisions of the embryo, but its origin remains elusive. Human zygotes that cluster their nucleoli at the pronuclear interface are thought to be more likely to develop into healthy euploid embryos. Here, we show that the parental genomes cluster with nucleoli in each pronucleus within human and bovine zygotes, and clustering is required for the reliable unification of the parental genomes after fertilization. During migration of intact pronuclei, the parental genomes polarize toward each other in a process driven by centrosomes, dynein, microtubules, and nuclear pore complexes. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in direct proximity to each other, yet separated. Parental genome clustering ensures the rapid unification of the parental genomes on nuclear envelope breakdown. However, clustering often fails, leading to chromosome segregation errors and micronuclei, incompatible with healthy embryo development., Graphical abstract, Highlights • The parental genomes cluster at the pronuclear interface in human and bovine zygotes • Clustering is driven by centrosomes, which often reside at the pronuclear interface • Dynein orients chromosomes toward centrosomes via nuclear pore complexes as adaptors • Clustering defects lead to aneuploidy and micronuclei, impairing embryo development, In human and bovine zygotes, parental genomes cluster and polarize toward each other in a highly error-prone process driven by centrosomes, dynein, microtubules, and nuclear pore complexes. Failure to cluster the parental genomes leads to chromosome segregation errors and micronuclei, which are incompatible with healthy embryo development.

Methylation is a universal biochemical process which covalently adds methyl groups to a variety of molecular targets. It plays a critical role in two major global regulatory mechanisms, epigenetic modifications and imprinting, via methyl tagging on histones and DNA. During reproduction, the two genomes that unite to create a new individual are complementary but not equivalent. Methylation determines the complementary regulatory characteristics of male and female genomes. DNA methylation is executed by methyltransferases that transfer a methyl group from S-adenosylmethionine, the universal methyl donor, to cytosine residues of CG (also designated CpG). Histones are methylated mainly on lysine and arginine residues. The methylation processes regulate the main steps in reproductive physiology: gametogenesis, and early and late embryo development. A focus will be made on the impact of assisted reproductive technology and on the impact of endocrine disruptors (EDCs) via generation of oxidative stress.

Summary Chromosome segregation errors during female meiosis are a leading cause of pregnancy loss and human infertility. The segregation of chromosomes is driven by interactions between spindle microtubules and kinetochores. Kinetochores in mammalian oocytes are subjected to special challenges: they need to withstand microtubule pulling forces over multiple hours and are built on centromeric chromatin that in humans is decades old. In meiosis I, sister kinetochores are paired and oriented toward the same spindle pole. It is well established that they progressively separate from each other with advancing female age. However, whether aging also affects the internal architecture of centromeres and kinetochores is currently unclear. Here, we used super-resolution microscopy to study meiotic centromere and kinetochore organization in metaphase-II-arrested eggs from three mammalian species, including humans. We found that centromeric chromatin decompacts with advancing maternal age. Kinetochores built on decompacted centromeres frequently lost their integrity and fragmented into multiple lobes. Fragmentation extended across inner and outer kinetochore regions and affected over 30% of metaphase-II-arrested (MII) kinetochores in aged women and mice, making the lobular architecture a prominent feature of the female meiotic kinetochore. We demonstrate that a partial cohesin loss, as is known to occur in oocytes with advancing maternal age, is sufficient to trigger centromere decompaction and kinetochore fragmentation. Microtubule pulling forces further enhanced the fragmentation and shaped the arrangement of kinetochore lobes. Fragmented kinetochores were frequently abnormally attached to spindle microtubules, suggesting that kinetochore fragmentation could contribute to the maternal age effect in mammalian eggs., Graphical Abstract, Highlights • Centromeric chromatin in oocytes decompacts as females age • MI/MII kinetochores built on decompacted centromeres fragment into multiple lobes • A partial loss of cohesin is sufficient to mimic these changes in young eggs • Fragmented kinetochores frequently interact incorrectly with spindle microtubules, Aneuploidy in eggs increases dramatically as females age. Zielinska et al. show that aging affects the internal architecture of centromeres and kinetochores in mammalian eggs, including humans. As cohesin is lost, centromeric chromatin decompacts and kinetochores fragment, which is linked to incorrect chromosome-microtubule attachments.

This paper reports on the numbers of treatment cycles involved in the development of IVF (1969-1978) and their outcomes. We show that between 1969 and 1978: (i) a minimum of 282 women were involved in 495 cycles of potential laparoscopic oocyte retrieval (LOR); (ii) of these cycles, 457/495 proceeded to LOR to attempt egg collection; (iii) of which an outcome was recorded in 436/457; (iv) eggs were recovered in 388/436 of these; (v) inseminations were recorded in 331/388; (vi) embryos were recorded in at least 167; (vii) a total of 112 embryo transfers were attempted; and (viii) a maximum of 11 possible biochemical/preclinical pregnancies plus five clinical pregnancies were observed; (ix) from which two healthy live births resulted.

Aneuploidy in human eggs is the leading cause of pregnancy loss and several genetic disorders such as Down syndrome. Most aneuploidy results from chromosome segregation errors during the meiotic divisions of an oocyte, the egg’s progenitor cell. The basis for particularly error-prone chromosome segregation in human oocytes is not known. We analyzed meiosis in more than 100 live human oocytes and identified an error-prone chromosome-mediated spindle assembly mechanism as a major contributor to chromosome segregation defects. Human oocytes assembled a meiotic spindle independently of either centrosomes or other microtubule organizing centers. Instead, spindle assembly was mediated by chromosomes and the small guanosine triphosphatase Ran in a process requiring ~16 hours. This unusually long spindle assembly period was marked by intrinsic spindle instability and abnormal kinetochore-microtubule attachments, which favor chromosome segregation errors and provide a possible explanation for high rates of aneuploidy in human eggs.

Three major sources of financial support for the research undertaken by Edwards, Steptoe and Purdy between 1969 and 1978 are identified: the Ford Foundation, Oldham and District General Hospital (ODGH) Management Committee, and Miss Lillian Lincoln Howell via the American Friends of Cambridge University. Significant possible financial support from the World Health Organization was also identified. In addition, evidence of support in kind from GD Searle and Co. plus staff at ODGH was found. Expenditure on salaries of staff at Oldham was negligible, as most volunteered their time outside of their official paid duties. Work in Cambridge was evidently funded largely from Ford Foundation grants, as was Edwards' salary and probably that of Purdy. Clinical costs seem to have been largely borne by ODGH. The funds from Lillian Lincoln Howell supported travel and accommodation costs plus office costs. Overall, Edwards, Steptoe and Purdy achieved reasonable support for the programme of research, despite the initial rejection of funding by the Medical Research Council. However, this was at the expense of considerable inconvenience to Purdy and Edwards, and depended upon the good will of staff led by Muriel Harris in Oldham, who donated their time and expertise. As a result of our research, we conclude that, to Edwards, Steptoe and Purdy, should be added the names of two other hitherto neglected people who were essential to the success of this pioneering research: namely Muriel Harris and Lillian Lincoln Howell. Keywords: Ford Foundation, IVF expenditure and grants, Lillian Lincoln Howell, Muriel Harris, Oldham and District General Hospital

The role of Jean Purdy in the work leading to the birth of Louise Brown is assessed. We report that Purdy: (i) recorded and organized most of the data systematically; (ii) probably spent longer working in Oldham than did Edwards; (iii) whilst there, was primarily responsible for organizing laboratory supplies, including media preparation and testing; (iv) was involved in patient care; and (v) was a major source of support to Edwards. We find that Purdy, despite her nursing qualification, was not involved in laparoscopic egg retrieval and clinical aspects, but was focused on basic research activities. The evidence on who was present at embryo transfers is less clear, but suggests that Edwards was present for all, whereas Purdy may have been absent for some. Overall, we conclude that Purdy’s role was a highly significant and under-appreciated element in the achievement of IVF in Oldham. Keywords: Edwards and Steptoe, IVF, Louise Brown, Oldham, Purdy

A survey is presented of the various technical and scientific challenges that had to be met during the 10-year period before the first successful live birth after IVF and embryo transfer was achieved, and the approaches used to meet these challenges is discussed. Records dated from January 1969 to July 1978 indicate that a minimum of 282 women were involved in 495 cycles scheduled for laparoscopic oocyte recovery, of which 457 cycles (92%) proceeded to attempted egg collection. A total of 1361 eggs were recovered over 388 cycles, of which 1237 (91%) are recorded as having been inseminated in 331 (85%) of these cycles. Approximately 221 embryos were described in 165 (43%) of the 388 cycles. A total of 112 embryo transfers were attempted, which resulted in five clinical pregnancies with two live births. This paper discusses the ways in which hormonal stimulation of follicle growth to the pre-ovulatory stage was varied, and the endocrine monitoring of these variations in blood, urine and follicular fluid, as well as their influence on egg recovery and fertilization rates. Variations in media composition and preparation are also described. It is concluded that, whilst driven by scientific reasoning, the approach adopted in trying to achieve successful IVF was empirical rather than evidence-driven. Keywords: blastocyst development, early pregnancy, human embryo culture, luteal phase abnormalities, natural cycles, pregnancy losses, stimulated follicular maturation

This paper reports on the numbers of treatment cycles involved in the development of IVF (1969–1978) and their outcomes. We show that between 1969 and 1978: (i) a minimum of 282 women were involved in 495 cycles of potential laparoscopic oocyte retrieval (LOR); (ii) of these cycles, 457/495 proceeded to LOR to attempt egg collection; (iii) of which an outcome was recorded in 436/457; (iv) eggs were recovered in 388/436 of these; (v) inseminations were recorded in 331/388; (vi) embryos were recorded in at least 167; (vii) a total of 112 embryo transfers were attempted; and (viii) a maximum of 11 possible biochemical/preclinical pregnancies plus five clinical pregnancies were observed; (ix) from which two healthy live births resulted. Keywords: embryo transfers, fertilisation rates, history of IVF, laparoscopic oocyte retrieval, pregnancy outcomes

The oviduct has long been considered a 'pipeline', a tube allowing transit of spermatozoa and embryos; this perspective has been reinforced by the success of human IVF. Evidence accumulated over several decades, however, indicates that embryos can modulate the metabolism of tubal cells in their environment. Human IVF culture media is based on formulations that pass mouse embryo assays as quality control: the requirements of mouse embryos differ from those of human embryos, and therefore conditions for human IVF are far removed from the natural environment of the oviduct. The preimplantation environment, both in vitro and in vivo, is known to affect the health of offspring through mechanisms that influence imprinting. Recent studies also show that male accessory glands act in synergy with the oviduct in providing an optimal environment, and this represents a further perspective on the oviduct's contribution to harmonious embryo development and subsequent long-term health. The metabolism of the human embryo is far from being understood, and a 'return' to in-vivo conditions for preimplantation development is worthy of consideration. Although results obtained in rodents must be interpreted with caution, lessons learned from animal embryo culture must not be neglected.

Purpose Gametocyte-specific factor 1 has been shown in other species to be required for the silencing of retrotransposons via the Piwi-interacting RNA (piRNA) pathway. In this study, we aimed to isolate and assess expression of transcripts of the gametocyte-specific factor 1 (GTSF1) gene in the human female germline and in preimplantation embryos. Methods Complementary DNA (cDNA) libraries from human fetal ovaries and testes, human oocytes and preimplantation embryos and ovarian follicles isolated from an adult ovarian cortex biopsy were used to as templates for PCR, cloning and sequencing, and real time PCR experiments of GTSF1 expression. Results GTSF1 cDNA clones that covered the entire coding region were isolated from human oocytes and preimplantation embryos. GTSF1 mRNA expression was detected in archived cDNAs from staged human ovarian follicles, germinal vesicle (GV) stage oocytes, metaphase II oocytes, and morula and blastocyst stage preimplantation embryos. Within the adult female germline, expression was highest in GV oocytes. GTSF1 mRNA expression was also assessed in human fetal ovary and was observed to increase during gestation, from 8 to 21 weeks, during which time oogonia enter meiosis and primordial follicle formation first occurs. In human fetal testis, GTSF1 expression also increased from 8 to 19 weeks. Conclusions To our knowledge, this report is the first to describe the expression of the human GTSF1 gene in human gametes and preimplantation embryos. Electronic supplementary material The online version of this article (doi:10.1007/s10815-016-0795-0) contains supplementary material, which is available to authorized users.

The negative effect of oxidative stress on the human reproductive process is no longer a matter for debate. Oxidative stress affects female and male gametes and the developmental capacity of embryos. Its effect can continue through late stages of pregnancy. Metabolic disorders and psychiatric problems can also be caued by DNA methylation and epigenetic errors. Age has a negative effect on oxidative stress and DNA methylation, and recent observations suggest that older men are at risk of transmitting epigenetic disorders to their offspring. Environmental endocrine disruptors can also increase oxidative stress and methylation errors. Oxidative stress and DNA methylation feature a common denominator: the one carbon cycle. This important metabolic pathway stimulates glutathione synthesis and recycles homocysteine, a molecule that interferes with the process of methylation. Glutathione plays a pivotal role during oocyte activation, protecting against reactive oxygen species. Assisted reproductive techniques may exacerbate defects in methylation and epigenesis. Antioxidant supplements are proposed to reduce the risk of potentially harmful effects, but their use has failed to prevent problems and may sometimes be detrimental. New concepts reveal a significant correlation between oxidative stress, methylation processes and epigenesis, and have led to changes in media composition with positive preliminary clinical consequences.

There is evidence that expression and methylation of the imprinted paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) gene may be affected by assisted reproductive technologies (ARTs) and infertility. In this study, we sought to assess the imprinting status of the MEST gene in a large cohort of in vitro-derived human preimplantation embryos, in order to characterise potentially adverse effects of ART and infertility on this locus in early human development. Embryonic genomic DNA from morula or blastocyst stage embryos was screened for a transcribed AflIII polymorphism in MEST and imprinting analysis was then performed in cDNA libraries derived from these embryos. In 10 heterozygous embryos, MEST expression was monoallelic in seven embryos, predominantly monoallelic in two embryos, and biallelic in one embryo. Screening of cDNA derived from 61 additional human preimplantation embryos, for which DNA for genotyping was unavailable, identified eight embryos with expression originating from both alleles (biallelic or predominantly monoallelic). In some embryos, therefore, the onset of imprinted MEST expression occurs during late preimplantation development. Variability in MEST imprinting was observed in both in vitro fertilization and intracytoplasmic sperm injection-derived embryos. Biallelic or predominantly monoallelic MEST expression was not associated with any one cause of infertility. Characterisation of the main MEST isoforms revealed that isoform 2 was detected in early development and was itself variably imprinted between embryos. To our knowledge, this report constitutes the largest expression study to date of genomic imprinting in human preimplantation embryos and reveals that for some imprinted genes, contrasting imprinting states exist between embryos.

Mouse embryos segregate three different lineages during preimplantation development: trophoblast, epiblast and hypoblast. These differentiation processes are associated with restricted expression of key transcription factors (Cdx2, Oct4, Nanog and Gata6). The mechanisms of segregation have been extensively studied in the mouse, but are not as well characterised in other species. In the human embryo, hypoblast differentiation has not previously been characterised. Here we demonstrate co-exclusive immunolocalisation of Nanog and Gata4 in human blastocysts, implying segregation of epiblast and hypoblast, as in rodent embryos. However, the formation of hypoblast in the human is apparently not dependent upon FGF signalling, in contrast to rodent embryos. Nonetheless, the persistence of Nanog-positive cells in embryos following treatment with FGF inhibitors is suggestive of a transient naïve pluripotent population in the human blastocyst, which may be similar to rodent epiblast and ES cells but is not sustained during conventional human ES cell derivation protocols., Highlights ► Segregation of epiblast and Gata4-positive hypoblast in human blastocysts ► Insensitivity of human hypoblast formation to FGF/Erk inhibition ► Persistence of Nanog-positive cells in human epiblast after FGF/Erk inhibition

The steep decline in both natural fertility and success after assisted reproduction treatment with increasing maternal age is universally recognized. Large variations in the developmental competence of oocytes collected are seen during assisted cycles, and a link between the biological competence of oocytes retrieved and age has been confirmed. Patients who require donated oocytes can benefit from egg sharing programmes, in which a proportion of oocytes collected from selected patients aged ⩽35years undergoing conventional assisted reproduction treatment are shared with a matched recipient. The reproductive outcomes of the egg provider and recipient can thus be compared to quantify the significance of oocyte quality. Data gathered from two comparable treatment centres resulted in 285 pairs of egg sharing providers and their recipients that could be analysed statistically. The chief finding was donor pregnancy as a predictor of recipient pregnancy given embryo transfer (odds ratio 2.15, 95% confidence interval 1.33–3.46, P ≈0.002), despite an appreciably higher mean age of the recipients. The probability of a recipient pregnancy increased by almost 0.2. Such results strongly indicate the key importance of oocyte quality for a successful clinical outcome in egg sharing practices and assisted reproduction treatment more generally. Both natural fertility and success after assisted conception treatment are known to decline dramatically with increasing age. Oocytes collected during assisted reproduction cycles often show large variations in their ability to be fertilized and develop into viable embryos that will lead to a pregnancy after transfer, and a link between this biological competence and age has been confirmed. A major source of data lies in the study of egg donation programmes, in which oocytes retrieved from younger women are donated to recipients of an older age group. Egg sharing is a type of egg donation treatment in which a proportion of oocytes collected from selected patients aged ⩽35years of age undergoing conventional assisted reproduction treatment are shared with a matched recipient whose ovaries are no longer able to produce viable oocytes. The treatment outcomes of the egg provider and recipient can thus be compared as an indicator of oocyte quality. In this study, data from two comparable treatment centres were combined to yield results regarding the treatment outcome for 285 pairs of egg sharing providers and their recipients. Statistical analysis revealed that when both donor and recipient achieved embryo transfer, a donor pregnancy significantly increased the chances of her recipient also achieving a pregnancy, despite the fact that the recipients were of an appreciably older age group. Such results strongly indicate that oocyte quality is of key importance for a successful clinical outcome in egg sharing practices and assisted reproduction treatment more generally.

Androgens play key roles in spermatogenesis, and they exert their effect via the androgen receptor (AR). The AR gene has a repetitive DNA sequence in exon 1 that encodes a polyglutamine tract. Instability in the glutamine (CAG) repeat unit length is polymorphic across ethnic groups. Previous studies of the relationship between the repeat unit length and male infertility have been contradictory. To establish the range of wild-type alleles in Egyptian men, we determined the range of repeat lengths in a population of normally fertile, ethnically selected Egyptian men. We also investigated the association between trinucleotide repeat length within the AR gene and male factor infertility in a population of ethnically selected Egyptian infertile men, who were compared with fertile, ethnic group-matched and age-matched controls. The study included 129 clinically selected infertile Egyptian men who were scheduled for intracytoplasmic sperm injection and 52 ethnically matched fertile controls. The experimental population was grouped according to sperm counts ranging from nonobstructive azoospermia to normozoospermia. The CAG repeat N-terminal domain region of the AR gene was amplified in peripheral blood DNA, and allele size was determined by fragment analysis. Allele size and single-nucleotide polymorphism and mutation rates were determined by sequencing individual amplified alleles. The mean CAG repeat length in the azoospermia group was 18.55 +/- 2.0; in the severe oligozoospermia group it was 18.21 +/- 3.42; in the oligozoospermia group it was 18.27 +/- 2.93; and in the infertile with normal sperm count group it was 17.72 +/- 2.0. In the control group, the mean CAG repeat length was 18.18 +/- 3.63. No significant correlation was found between CAG repeat length and the risk of male factor infertility in an ethnically defined population of Egyptian men. However, a significant and positive correlation between CAG repeat length and serum testosterone concentration was demonstrated. This suggests the involvement of epigenetic regulation linked to this region.

Aneuploidy in human eggs is the leading cause of pregnancy loss and Down’s syndrome. Aneuploid eggs result from chromosome segregation errors when an egg develops from a progenitor cell, called an oocyte. The mechanisms that lead to an increase in aneuploidy with advanced maternal age are largely unclear. Here, we show that many sister kinetochores in human oocytes are separated and do not behave as a single functional unit during the first meiotic division. Having separated sister kinetochores allowed bivalents to rotate by 90 degrees on the spindle and increased the risk of merotelic kinetochore-microtubule attachments. Advanced maternal age led to an increase in sister kinetochore separation, rotated bivalents and merotelic attachments. Chromosome arm cohesion was weakened, and the fraction of bivalents that precociously dissociated into univalents was increased. Together, our data reveal multiple age-related changes in chromosome architecture that could explain why oocyte aneuploidy increases with advanced maternal age.

Maintaining consistent and reliably high success rates is a daily challenge for every IVF laboratory. This step-by-step guide is an essential aid in navigating the complex maze of physical, chemical, biological, and logistic parameters that underpin successful gamete and embryo culture: temperature, pH, osmolality, gas supplies, air quality, light exposure, infections, managing supplies, personnel, as well as overall quality control. Numerous real-life troubleshooting case reports are presented, identifying all aspects necessary for troubleshooting. Process maps and flow charts accompanying each chapter offer a logical and systematic approach to problem solving in the laboratory. This is an essential resource for scientists in assisted reproductive technology and specialists in reproductive biology and medicine, helping IVF clinics to achieve the dream of every infertile couple: the birth of a healthy child.