The Oldham Notebooks: an analysis of the development of IVF 1969-1978. I. Introduction, materials and methods.

In this introductory paper, we describe the primary source material studied in this Symposium, namely a set of 21 notebooks and 571 pages of loose sheets and scraps of paper, which, on cross-referencing, have allowed us to reconstruct the sequence, timing and numbers of the laparoscopic cycles planned, attempted and undertaken between 9 January 1969 and 1 August 1978 by Robert Edwards, Patrick Steptoe and Jean Purdy in Oldham, UK, as well as to identify most of the patients involved. In addition, we describe the background to the five papers that follow, and the secondary sources and recorded interviews, which have provided useful ancillary material.The research was supported by grants from the Wellcome Trust (088708 to Nick Hopwood, Martin Johnson et al. and 094985 to Allen Packwood and Martin Johnson), which otherwise had no involvement in the research or its publication.This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S2405661815000027

The Oldham Notebooks: an analysis of the development of IVF 1969-1978. IV. Ethical aspects.

Six evidential sources are examined to investigate how Edwards and Steptoe applied ethical standards to their research leading to the birth of Louise Brown: (i) Their own contemporary writings from 1970 onwards. (ii) Archival evidence from the British Medical Association (BMA), the British Association for the Advancement of Science (BAAS), and correspondence between Edwards and the Ford Foundation. (iii) Minutes of Oldham General Hospital (OGH) Ethics Committee. (iv) Letters by Edwards to prospective patients. (v) oral evidence from interviews with a patient and colleagues. (vi) Evidence from their clinical case management of patients. Taken together these sources suggest that Edwards and Steptoe demonstrated a strong awareness of the ethical issues involved, and offer evidence of honesty to patients about the realistic prospects of success and ethical practice. Nonetheless, decisive evidence that ethical aspirations were put into practice is not available.The research was supported by grants from the Wellcome Trust (088708 and 094985), which otherwise had no involvement in the research or its publication.This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S2405661815000039

The Oldham Notebooks: an analysis of the development of IVF 1969-1978. II. The treatment cycles and their outcomes.

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.The research was supported by grants from the Wellcome Trust (088708 and 094985), which otherwise had no involvement in the research or its publication.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.rbms.2015.04.00

Link Between Increased Prevalence of Autism Spectrum Disorder Syndromes and Oxidative Stress, DNA Methylation, and Imprinting

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Ionized concentrations in Ca2+ and Mg2+ buffers must be measured, not calculated

Modelling intracellular regulation of Ca2+/Mg2+ is now an established part of physiology. However, the conclusions drawn from such studies depend on accurate knowledge of intracellular [Ca2+], ∆[Ca2+] and the pK′ values for the intracellular binding of Ca2+/Mg2+. Calculation of [Ca2+]/[Mg2+] in buffers is normal. The eight freely available programs all give different values for the [Ca2+]/[Mg2+] in the buffer solutions, varying by up to a factor of 4.3. As a result, concentrations must be measured. There are two methods to do this, both based on the ligand optimization method (LOM): (1) calibration solutions from 0.5 to 4 mmol l−1; and (2) calibration solutions from 0.1 µmol l−1 to 2 mmol l−1. Both methods can be used to calibrate Ca2+/Mg2+ electrodes. Only Method 2 can be used directly to calibrate fluorochromes and aequorin. Software in the statistical program R to calculate the [Ca2+]/[Mg2+] in buffers is provided for both methods. The LOM has now been generalized for use with electrodes, fluorochromes and aequorin, making it the ideal method to determine the pK′ values for intracellular binding of Ca2+/Mg2+. The [Ca2+]/[Mg2+] in buffers must be measured routinely, which is best done by calibrating electrodes with the LOM and software written in R. If [Ca2+]/[Mg2+] in buffers are calculated, the parameters used in modelling show the same degree of variability as the software programs. Uncritical acceptance of such parameters means that conclusions reached from such studies are relative, not absolute, and must now be re‐examined

Menstrual flow as a non-invasive source of endometrial organoids.

Assessment of the endometrium often necessitates a biopsy, which currently involves an invasive, transcervical procedure. Here, we present an alternative technique based on deriving organoids from menstrual flow. We demonstrate that organoids can be derived from gland fragments recovered from menstrual flow. To confirm they faithfully reflect the in vivo state we compared organoids derived from paired scratch biopsies and ensuing menstrual flow from patients undergoing in vitro fertilisation (IVF). We demonstrate that the two sets of organoids share the same transcriptome signature, derivation efficiency and proliferation rate. Furthermore, they respond similarly to sex steroids and early-pregnancy hormones, with changes in morphology, receptor expression, and production of 'uterine milk' proteins that mimic those during the late-secretory phase and early pregnancy. This technique has wide-ranging impact for non-invasive investigation and personalised approaches to treatment of common gynaecological conditions, such as endometriosis, and reproductive disorders, including failed implantation after IVF and recurrent miscarriage

MICA: a multi-omics method to predict gene regulatory networks in early human embryos

Recent advances in single-cell omics have transformed characterisation of cell types in challenging-to-study biological contexts. In contexts with limited single-cell samples, such as the early human embryo inference of transcription factor-gene regulatory network (GRN) interactions is especially difficult. Here, we assessed application of different linear or non-linear GRN predictions to single-cell simulated and human embryo transcriptome datasets. We also compared how expression normalisation impacts on GRN predictions, finding that transcripts per million reads outperformed alternative methods. GRN inferences were more reproducible using a non-linear method based on mutual information (MI) applied to single-cell transcriptome datasets refined with chromatin accessibility (CA) (called MICA), compared with alternative network prediction methods tested. MICA captures complex non-monotonic dependencies and feedback loops. Using MICA, we generated the first GRN inferences in early human development. MICA predicted co-localisation of the AP-1 transcription factor subunit proto-oncogene JUND and the TFAP2C transcription factor AP-2γ in early human embryos. Overall, our comparative analysis of GRN prediction methods defines a pipeline that can be applied to single-cell multi-omics datasets in especially challenging contexts to infer interactions between transcription factor expression and target gene regulation

Menstrual flow as a non-invasive source of endometrial organoids

Funder: Centre for Trophoblast ResearchAbstract: Assessment of the endometrium often necessitates a biopsy, which currently involves an invasive, transcervical procedure. Here, we present an alternative technique based on deriving organoids from menstrual flow. We demonstrate that organoids can be derived from gland fragments recovered from menstrual flow. To confirm they faithfully reflect the in vivo state we compared organoids derived from paired scratch biopsies and ensuing menstrual flow from patients undergoing in vitro fertilisation (IVF). We demonstrate that the two sets of organoids share the same transcriptome signature, derivation efficiency and proliferation rate. Furthermore, they respond similarly to sex steroids and early-pregnancy hormones, with changes in morphology, receptor expression, and production of ‘uterine milk’ proteins that mimic those during the late-secretory phase and early pregnancy. This technique has wide-ranging impact for non-invasive investigation and personalised approaches to treatment of common gynaecological conditions, such as endometriosis, and reproductive disorders, including failed implantation after IVF and recurrent miscarriage

A single cell characterisation of human embryogenesis identifies pluripotency transitions and putative anterior hypoblast centre.

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