RESEARCH QUESTION:What implications for policy and practice can be derived from outcomes and trends observed across 8 years of a surrogacy programme in two UK-regulated IVF centres (London, Cardiff)?DESIGN:Retrospective cohort study analysing surrogacy treatments undertaken between 2014 and September 2021.RESULTS:Surrogacy continues to rise in popularity in the UK despite the inability of those supporting safe and professional practice to advertise to recruit surrogates. In two IVF centres regulated by the Human Fertilisation and Embryology Authority (HFEA), both the number of surrogacy treatments and the proportion of those undertaken on behalf of same-sex male intended parents increased year on year in the period studied. From a cohort of 108 surrogates, 71 babies were born to 61 surrogates (with five pregnancies ongoing) by February 2022. No statistically significant difference in live birth rates (LBR) was observed between the heterosexual couples and same-sex male couples. Sample sizes of single and transgender intended parents were too small (n < 5) to compare. The use of vitrified oocytes in surrogacy treatments has increased year on year, while fresh oocyte use has declined since peaking in 2019. There was no significant difference in LBR between fresh and vitrified oocyte usage across the cohort.CONCLUSIONS:The number of surrogacy treatments steadily increased, with clear evidence that the proportion of same-sex male couples accessing surrogacy is a major contributor to this growth. Vitrified/warmed oocyte use now outstrips the use of fresh oocytes in the surrogacy treatment cycles studied here. The results represent a strong basis for supporting the liberalization of regulatory reform expected to be introduced in the UK later in 2022.

The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

This study describes the use of an automated measurement of follicular diameter using the new Sono-Automatic Volume Calculation (Sono-AVC) software and compares the accuracy of automated measures and the time taken for such measures to those made manually from two-dimensional and three-dimensional ultrasound. Sono-AVC provides measurements of follicular diameter that are more accurate than the manual measures and has the potential to improve the clinical work flow because the time taken for the measurements is significantly shorter. (Fertil Steril (R), 2009;91:1469-72. (C)2009 by American Society for Reproductive Medicine.)

Research into the behavior, efficacy, and biosafety of stem cells with a view to clinical transplantation requires the development of noninvasive methods for in vivo imaging of cells transplanted into animal models. This is particularly relevant for human embryonic stem cells (hESCs), because transplantation of undifferentiated hESCs leads to tumor formation. The present study aimed to monitor hESCs in real time when injected in vivo. hESCs were stably transfected to express luciferase, and luciferase expression was clearly detected in the undifferentiated and differentiated state. When transfected hESCs were injected into chick embryos, bioluminescence could be detected both ex and in ovo. In the SCID mouse model, undifferentiated hESCs were detectable after injection either into the muscle layer of the peritoneum or the kidney capsule. Tumors became detectable between days 10-30, with approximately a 3 log increase in the luminescence signal by day 75. The growth phase occurred earlier in the kidney capsule and then reached a plateau, whilst tumors in the peritoneal wall grew steadily throughout the period analysed. These results show the widespread utility of bioluminescent for in vivo imaging of hESCs in a variety of model systems for preclinical research into regenerative medicine and cancer biology.

call for careful preclinical validation to find the optimal system in each laboratory setting.Regarding legislation, open or closed systems perform equally well and choice will depend on severity of implementation of EU directives in EU member states.

Human embryonic stem cells (hESCs) are thought to be susceptible to chromosomal rearrangements as a consequence of single cell dissociation. Compared in this study are two methods of dissociation that do not generate single cell suspensions (collagenase and EDTA) with an enzymatic procedure using trypsin combined with the calcium-specific chelator EGTA (TEG), that does generate a single cell suspension, over 10 passages. Cells passaged by single cell dissociation using TEG retained a normal karyotype. However, cells passaged using EDTA, without trypsin, acquired an isochromosome p7 in three replicates of one experiment. In all of the TEG, collagenase and EDTA-treated cultures, cells retained consistent telomere length and potentiality, demonstrating that single cell dissociation can be used to maintain karyotypically and phenotypically normal hESCs. However, competitive genomic hybridization revealed that subkaryotypic deletions and amplifications could accumulate over time, reinforcing that present culture regimes remain suboptimal. In all cultures the cell surface marker CD30, reportedly expressed on embryonal carcinoma but not karyoptically normal ESCs, was expressed on hESCs with both normal and abnormal karyotype, but was upregulated on the latter.

Objectives To assess the ability of the new software SonoAVC to measure follicular volume and to compare these volume calculations with those made by conventional methods.Methods Three-dimensional ultrasound imaging was used to acquire volumetric data from the ovaries of 51 women undergoing controlled ovarian stimulation as part of in-vitro fertilization treatment. All assessments were performed on the day of oocyte retrieval and the true volume of each follicle was ascertained by manual measurement of the follicular aspirate. SonoAVC was used to automatically measure the volume of follicles and to provide three perpendicular diameters (xyz diameters), which were used to estimate volume using the sphere formula. The sphere formula was also used to estimate the volume from manual measurements of follicle diameter derived from conventional two-dimensional (2D) displays. Virtual Organ Computer-aided AnaLysis (VOCAL) was also used to measure volume, and the validity of each technique was compared using limits of agreement.Results Two hundred and twenty-four follicles with a mean follicular volume of 3.7 (range, 0.4-16.2) cm(3) were studied. SonoAVC provided highly accurate automatic follicular volume measurements in all cases. Volume estimations made from the automatic maximal follicular diameter measurements (xyz diameters) were less valid. VOCAL proved highly valid but was less accurate than SonoAVC. Volumes estimated from manually derived follicular diameter measurements were the least accurate.Conclusions SonoAVC provides highly valid, automatic measurements of follicular volume. These measurements are more accurate than volumes estimated from 2D manual measurements, automated measurements of follicular diameter and those calculated using VOCAL. Copyright (C) 2008 ISUOG. Published by John Wiley & Sons, Ltd.

Although undifferentiated human embryonic stem cells (hESCs) are tumorigenic, this capacity is lost after differentiation, and hESCs are being widely investigated for applications in regenerative medicine. To engineer protection against the unintentional transplantation of undifferentiated cells, we generated hESCs carrying a construct in which the alpha 1,3-galactosyltransferase (GalT) open reading frame was transcribed from the hTERT promoter (pmGT). Because the endogenous GalT gene is inactive, GalT expression was limited to undifferentiated cells. A second chimeric construct (pmfGT) differed by replacement of the GalT leader sequence for that of the fucosyltransferase gene. Two subclones containing stable integrations of pmGT and pmfGT (M2 and F11, respectively) were assessed for their response to human serum containing antibodies to the Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-gal) epitope. The low-variegation line, M2, and to a lesser extent the more variegated line F11, were sensitive to human serum when exposed in the undifferentiated state. However, M2 cells were largely insensitive after differentiation and retained both a normal karyotype and the ability to differentiate into derivatives of the three germ layers in severe combined immunodeficient mice. These data exemplify a method of protection against residual, undifferentiated hESCs prior to engraftment and may provide ongoing immune surveillance after engraftment against dedifferentiation or against de novo tumorigenesis involving hTERT reactivation. Untransfected H9 cells were not sensitive to the human serum used in this study. Hence, in our system, interactions of hESCs with other circulating antibodies, such as anti-Neu5Gc, were not observed.

Widespread provision of human embryonic stem cells (hESCs) for therapeutic use, drug screening and disease modelling will require cell lines sustainable over long periods in culture. Since the short-term, in vitro culture of mammalian embryos can result in DNA methylation changes, the epigenetic stability of hESCs warrants investigation. Existing hESC lines have been derived and cultured under diverse conditions, providing the potential for programming differential changes into the epigenome that may result in inter-line variability over and above that inherited from the embryo. By examining the DNA methylation profiles of > 2000 genomic loci by Restriction Landmark Genome Scanning, we identified substantial inter-line epigenetic distance between six independently derived hESC lines. Lines were found to inherit further epigenetic changes over time in culture, with most changes arising in the earliest stages post-derivation. The loci affected varied between lines. The majority of culture-induced changes (82.3-87.5%) were stably inherited both within the undifferentiated cells and post-differentiation. Adapting a line to a serum-free culture system resulted in additional epigenetic instability. Overall 80.5% of the unstable loci uncovered in hESCs have been associated previously with an adult tumour phenotype. Our study shows that current methods of hESC propagation can rapidly programme stable and unpredictable epigenetic changes in the stem cell genome. This highlights the need for (i) novel screening strategies to determine the experimental utility and biosafety of hESCs and (ii) optimization and standardization of procedures for the derivation and culture of hESC lines that minimize culture-induced instability.

Disregulation of imprinted genes can be associated with tumorigenesis and altered cell differentiation capacity and so could provide adverse outcomes for stem cell applications. Although the maintenance of mouse and primate embryonic stem cells in a pluripotent state has been reported to disrupt the monoallelic expression of several imprinted genes, available data have suggested relatively higher imprint stability in the human equivalents. Identification of 202 heterozygous loci allowed us to examine the allelic expression of 22 imprinted genes in 22 human embryonic stem cell lines. Half of the genes examined ( IPW , H19 , MEG3 , MEST isoforms 1 and 2, PEG10 , MESTIT1 , NESP55 , ATP10A , PHLDA2 , IGF2 ) showed variable allelic expression between lines, indicating vulnerability to disrupted imprinting. However, seven genes showed consistent monoallelic expression ( NDN , MAGEL2 , SNRPN , PEG3 , KCNQ1 , KCNQ1OT1 , CDKN1C ). Furthermore, four genes known to be monoallelic or to exhibit polymorphic imprinting in later-developing human tissues ( TP73 , IGF2R , WT1 , SLC22A18 ) were always biallelic in hESCs. MEST isoform 1, PEG10 , and NESP55 showed an association between the variability observed in interline allelic expression status and the DNA methylation of previously identified regulatory regions. Our results demonstrate gene-specific differences in the stability of imprinted loci in human embryonic stem cells and identify disrupted DNA methylation as one potential mechanism. We conclude the prudence of including comprehensive imprinting analysis in the continued characterization of human embryonic stem cell lines.

To assess the validity of manual measurements of follicular diameter with different 2D and 3D techniques and to compare these to measures derived from recently developed software (GE Healthcare) that automatically calculates the volume and relative dimensions of follicles within a 3D ultrasound dataset. A series of different sized follicles were chosen to provide a range of follicles with mean diameters of 11 to 22 mm. Five different follicles were examined within each size range. The true volume of each follicle was ascertained by manual measurement of the follicular aspirate and the estimated mean follicular diameter was calculated using the sphere formula. The mean diameter of each follicle was measured manually using a series of 2D and 3D techniques. The new software was used to provide an automatic measurement of the mean diameter (‘relaxed sphere diameter’) and three perpendicular diameters (‘xyz diameter’) of the follicle. Limits of agreement (LOA) were used to examine the difference in measurements. The ‘relaxed sphere diameter’ calculated by the automated software provided the most accurate measurements of follicular size, with values almost identical to the estimated diameter as calculated from the true follicular volume (Table). Automated ‘xyz’ measurements were less accurate and in many cases manual measurements were closer to the true value. Surprisingly the ‘single best estimate’ from a 2D image was the second most accurate technique although the LOA were better for measurements using the 3D display, especially for follicles measuring ≥ 18 mm. Recently developed software that automatically calculates the mean follicular diameter from 3D volumetric data provides a highly accurate assessment of follicle size. Manual measures are less accurate. The range of measurements is reduced when the 3D multiplanar view is used whilst the mean diameter and mean difference is not significantly different.

To assess the validity of new software (GE Healthcare) which automatically calculates the volume and dimensions of follicles within a 3D ultrasound dataset. 3D ultrasound was used to acquire volumetric data from the ovaries of 24 women undergoing ovarian stimulation as part of IVF treatment. All assessments were performed on the day of oocyte retrieval and the true volume of each follicle ascertained by manual measurement of the follicular aspirate. The new software automatically measures the volume of follicles and provides an estimate of the mean diameter (‘relaxed sphere diameter’) and the three perpendicular diameters (‘xyz diameters’). The mean diameter (MD) of these follicles was also measured manually using conventional 2D and 3D image displays and these values were used to estimate follicular volume (FV) using the sphere formula where FV (mL) = 4.1888 × (MD/2 cm)3. Limits of agreement were used to examine the validity of measurements. Some 100 follicles with a mean volume of 3.89 (range, 0.4–16.2) mL and diameter of 18.3 (range, 10.75–31.13) mm were studied. Results are summarized in the table. The software provided highly accurate automatic follicular volume measurements in all cases. Volume estimations made from automatic follicular diameter measurements using the ‘relaxed sphere diameter’ were equally valid whilst those made from the ‘xyz diameters’ were less valid but more accurate than volume estimations from manually derived follicular diameters. Volume estimations from manually derived diameters were more valid when the 3D multiplanar view was used. The new software provides automatic measurements of follicular diameter and volume that are more reliable and accurate than those estimated from manually derived follicular measurements. The software was developed in association with K plus Competence Center (Advanced Computer Vision) and part funded by the K plus Program.

Abstract Although undifferentiated human embryonic stem cells (hESCs) are tumorigenic, this capacity is lost after differentiation, and hESCs are being widely investigated for applications in regenerative medicine. To engineer protection against the unintentional transplantation of undifferentiated cells, we generated hESCs carrying a construct in which the α1,3-galactosyltransferase (GalT) open reading frame was transcribed from the hTERT promoter (pmGT). Because the endogenous GalT gene is inactive, GalT expression was limited to undifferentiated cells. A second chimeric construct (pmfGT) differed by replacement of the GalT leader sequence for that of the fucosyltransferase gene. Two subclones containing stable integrations of pmGT and pmfGT (M2 and F11, respectively) were assessed for their response to human serum containing antibodies to the Galα1-3Galβ1-4GlcNAc-R (α-gal) epitope. The low-variegation line, M2, and to a lesser extent the more variegated line F11, were sensitive to human serum when exposed in the undifferentiated state. However, M2 cells were largely insensitive after differentiation and retained both a normal karyotype and the ability to differentiate into derivatives of the three germ layers in severe combined immunodeficient mice. These data exemplify a method of protection against residual, undifferentiated hESCs prior to engraftment and may provide ongoing immune surveillance after engraftment against dedifferentiation or against de novo tumorigenesis involving hTERT reactivation. Untransfected H9 cells were not sensitive to the human serum used in this study. Hence, in our system, interactions of hESCs with other circulating antibodies, such as anti-Neu5Gc, were not observed.

Although all human ESC (hESC) lines have similar morphology, express key pluripotency markers, and can differentiate toward primitive germ layers in vitro, the lineage-specific developmental potential may vary between individual lines. In the current study, four hESC lines were cultured in the same feeder-free conditions to provide a standardized platform for interline analysis. A high-throughput, forced-aggregation system involving centrifugation of defined numbers of hESCs in V-96 plates (V-96FA) was developed to examine formation, growth, and subsequent cardiomyocyte differentiation from >22,000 EBs. Homogeneity of EBs formed by V-96FA in mouse embryo fibroblast-conditioned medium was significantly improved compared with formation in mass culture (p < .02; Levene's test). V-96FA EB formation was successful in all four lines, although significant differences in EB growth were observed during the first 6 days of differentiation (p = .044 to .001; one-way analysis of variance [ANOVA]). Cardiomyocyte differentiation potential also varied; 9.5% +/- 0.9%, 6.6% +/- 2.4%, 5.2% +/- 3.1%, and 1.6% +/- 1.0% beating EBs were identified for HUES-7, NOTT2, NOTT1, and BG01, respectively (p = .008; one-way ANOVA). Formation of HUES-7 V-96FA EBs in defined medium containing activin A and basic fibroblast growth factor resulted in 23.6% +/- 3.6% beating EBs, representing a 13.1-fold increase relative to mass culture (1.8% +/- 0.7%), consistent with an observed 14.8-fold increase in MYH6 (alphaMHC) expression by real-time polymerase chain reaction. In contrast, no beating areas were derived from NOTT1-EBs and BG01-EBs formed in defined medium. Thus, the V-96FA system highlighted interline variability in EB growth and cardiomyocyte differentiation but, under the test conditions described, identified HUES-7 as a line that can respond to cardiomyogenic stimulation.

The multipotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source of stem cells for transplant therapies and of vital importance given the shortage in organ donation. Recent studies suggest some immune privilege associated with hESC-derived tissues. However, the adaptability of the immune system makes it unlikely that fully differentiated tissues will permanently evade immune rejection. One promising solution is to induce a state of immune tolerance to a hESC line using tolerogenic hematopoietic cells derived from it. This could provide acceptance of other differentiated tissues from the same line. However, this approach will require efficient multilineage hematopoiesis from hESCs. This review proposes that more efficient differentiation of hESCs to the tolerogenic cell types required is most likely to occur through applying knowledge gained of the ontogeny of complex regulatory signals used by the embryo for definitive hematopoietic development in vivo. Stepwise formation of mesoderm, induction of definitive hematopoietic stem cells, and the application of factors key to their self-renewal may improve in vitro production both quantitatively and qualitatively.

Development of generic differentiation protocols that function in a range of independently-derived human embryonic stem cell (hESC) lines remains challenging due to considerable diversity in culture methods practiced between lines. Maintenance of BG01 and HUES-7 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual- and trypsin-passaging, respectively. We adapted both lines to trypsin-passaging on feeders or on Matrigel in feeder-free conditions and assessed proliferation and cardiac differentiation. On feeders, undifferentiated proliferation of BG01 and HUES-7 was supported by all three media tested (BG-SK, HUES-C and HUES-nL), although incidence of karyotypic instability increased in both lines in BG-SK. On Matrigel, KSR-containing conditioned medium (CM) promoted undifferentiated cell proliferation, while differentiation occurred in CM containing Plasmanate or ES-screened Fetal Bovine Serum (FBS) and in unconditioned medium containing 100 ng/ml bFGF. Matrigel cultures were advantageous for transfection but detrimental to embryoid body (EB) formation. However, transfer of hESCs from Matrigel back to feeders and culturing to confluence was found to rescue EB formation. EBs formed efficiently when hESCs on feeders were treated with collagenase, harvested by scraping and then cultured in suspension in CM. Subsequent culture in FBS-containing medium produced spontaneously contracting EBs, for which the mean beat rate was 37.2 +/- 2.3 and 41.1 +/- 3.1 beats / min for BG01-EBs and HUES-7-EBs, respectively. Derived cardiomyocytes expressed cardiac genes and responded to pharmacological stimulation. Therefore the same culture and differentiation conditions functioned in two independently-derived hESC lines. Similar studies in other lines may facilitate development of universal protocols.

To investigate a possible mechanism for inducing epigenetic defects in the preimplantation embryo, a human embryonic stem cell model was developed, and gene expression of the key methyl cycle enzymes, MAT2A, MAT2B, GNMT, SAHH, CBS, CGL, MTR, MTRR, BHMT, BHMT2, mSHMT, cSHMT and MTHFR was demonstrated, while MAT1 was barely detectable. Several potential acceptors of cycle-generated methyl groups, the DNA methyltransferases (DNMT1, DNMT3A, DNMT3B and DNMT3L), glycine methyltransferase and the polyamine biosynthetic enzymes, SAM decarboxylase and ornithine decarboxylase, were also expressed. Expression of folate receptor alpha suggests a propensity for folate metabolism. Methotrexate-induced depletion of folate resulted in elevated intracellular homocysteine concentration after 7 days in culture and a concomitant increase in cysteine and glutathione, indicating clearance of homocysteine through the transulphuration pathway. These studies indicate that altered methyl group metabolism provides a potential mechanism for inducing epigenetic changes in the preimplantation embryo.

Context The genetic code in the DNA of virtually every somatic cell can produce the entire complement of encoded proteins. Acetylation of histones and methylation of histones and DNA cytosine residues are part of the complex epigenetic regulatory process determining lineage-specific gene expression by altering the local structure of chromatin. After fertilisation, sperm DNA exchanges protamines for histones recruited from oocyte cytoplasm, reconfiguring both parental genomes into an epigenetic state conducive to activating the embryonic developmental programme. The identification of epigenetic reprogramming mechanisms is a major interest, rekindled by the ability of at least some somatic cells to acquire totipotency after somatic-cell nuclear transfer.Starting point Recently, Woo Suk Hwang and colleagues (Science 2004; 303: 1669-74) derived a human embryonic stem-cell line from embryo therapeutic cloning. Chad Cowan and colleagues (N Engl J Med 2004; 350: 1353-56) produced 17 new lines from embryos supernumerary to infertility treatments. However, increasing evidence from a range of mammals shows a propensity for epigenetic errors with embryo technologies. If paralleled in human embryos, the effect on tumorigenic and differentiation properties of embryonic stem cells needs to be established.Where next? Identifying the mechanisms in the oocyte that reprogramme a somatic cell to the embryonic state might allow somatic cells to be reprogrammed ex ovo by in-vitro manipulation of the epigenome. Because the oocyte is designed to reprogramme the sperm genome, which is in a different chromatin state from a somatic cell, perhaps many of the epigenetic errors induced by somatic-cell nuclear transfer could be avoided by a more targeted approach.