Vitrifying multiple embryos in different arrangements does not alter the cooling rate

Vitrification is the most common method of cryopreservation of gametes in fertility clinics due to its improved survival rates compared to slow freezing techniques. For the Open Cryotop® vitrification device, the number of oocytes, or embryos, mounted onto a single device can vary. In this work, a mathematical model is developed for the cooling of oocytes and embryos (samples). The model is solved computationally, to investigate whether varying the number of samples mounted onto the Open Cryotop® affects the cooling rates, and consequently the survival rates, of vitrified samples. Several realistic spatial arrangements of samples are examined, determining their temperature over time. In this way we quantify the effect of spatial arrangement on the cooling rate. Our results indicate that neither the spatial arrangement nor the number of mounted samples has a large effect on cooling rates, so long as the volume of the cryoprotectant remains minimal. The time taken for cooling is found to be on the order of half a second, or less, regardless of the spatial arrangement or number of mounted samples. Hence, rapid cooling can be achieved for any number or arrangement of samples, as long as device manufacturer guidelines are adhered to

First clinical report of 179 surrogacy cases in the UK: Implications for policy and practice

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 our HFEA-regulated IVF centres, both the number of surrogacy treatments and the proportion of those undertaken on behalf of same-sex male intended parents (IPs) increased year on year in the period studied. From our cohort of 108 surrogates, 71 babies were born to 61 surrogates (with 5 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 IPs and transgender IPs were too small (n<5) to compare. The use of vitrified oocytes in surrogacy treatments has increased year on year, whilst 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 in our clinics is steadily increasing, 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 our surrogacy treatment cycles. The results represent a strong basis for supporting liberalising regulatory reform expected to be introduced in the UK later this yea

The use of gene disruption to study the cytoskeletal proteins talin and vinculin.

Talin and vinculin are cytoskeletal proteins co-localising with the integrin family of adhesion receptors at sites of adhesion to the extracellular matrix. To investigate the role of these proteins in cell adhesion, gene disruption by homologous recombination has been used in an attempt to produce cells deficient in talin or vinculin. A C129 mouse embryonic stem (ES) cell ? library was screened with a 5' mouse talin cDNA and talin genomic clones isolated. A promoterless neo gene was inserted into the genomic DNA within the first two coding exons, forming a talin gene disruption vector. This vector failed to confer G418 resistance to ES cells, possibly due to the use of a neo gene containing a mutation which reduces the phosphotransferase activity of the encoded protein. A further vector was constructed using the same section of talin genomic DNA with a wild type neo gene driven by a PGK promoter. A tk cassette was included for selection against random integration. This vector was used successfully to isolate ES cells with one allele of the talin gene disrupted. However, talin levels were not reduced in these cells. This cell line will be useful to produce talin deficient cells or mice for the study of talin function. A vinculin gene targeting vector containing a tk negative selection marker and a neo gene driven by a PGK promoter was used unsuccessfully to disrupt the vinculin gene in ES cells. An alternative vector was constructed with a promoterless neo to reduce random integration, but this approach was also unsuccessful. The original vector used the mutant version of neo. The use of a disruption vector employing a wild type neo cassette has since led to the disruption of the vinculin gene. Wistar Furth rats have a point mutation (pro 1176 thr) in the talin gene. The mutation was introduced into a talin cDNA encoding residues 565-1328. The mutant and wild type cDNAs were expressed as GST-fusion proteins. The vinculin- and actin- binding activities of the expressed fusion proteins were assayed in vitro. The mutation did not prevent the binding of the talin polypeptide to vinculin or actin and the mutant polypeptide was still able to localise to focal adhesions when microinjected into fibroblasts

Occupational health issues experienced by UK embryologists: informing improvements in clinical reproductive science practice

A consultation exercise was undertaken with UK embryologists to construct knowledge of the occupational health issues they experience in everyday practice. Data were obtained from 223 eligible survey responses. Work-related ill health was self-reported by 58.3% of respondents, 76.2% of whom reported multiple issues. The most frequently disclosed ill-health conditions were musculoskeletal disorders (45.3%) and stress and mental health problems (27.8%). Other issues with an incidence above 3% were ocular and auditory problems and needlestick and liquid nitrogen injuries. Shoulder injury or pain correspondingly increased in incidence with length of time in service. Absence from work and/or light duties were necessitated for 34.5% of those affected. Assessment of the evidence base for these work-related ill-health conditions explored contributory and ameliorating factors, which enabled a series of evidence-based recommendations to be formulated via the adoption of a GRADE-based framework

Elevated expression of exogenous Rad51 leads to identical increases in gene-targeting frequency in murine embryonic stem (ES) cells with both functional and dysfunctional p53 genes

The Rad51 gene is the mammalian homologue of the bacterial RecA gene and catalyses homologous recombination in mammalian cells. In some cell types Rad51 has been shown to interact with p53, leading to inhibition of Rad51 activity. Here, we show a two- to four-fold increase in gene-targeting frequency at the HPRT locus using murine ES clones preengineered to overexpress Rad51, and a twofold increase in targeting frequency when a Rad51 expression cassette was cointroduced to wild-type ES cells with the targeting construct. In addition to its effect on homologous recombination, we show that Rad51 may down-regulate illegitimate recombination. We investigated the dependence of these phenomena upon p53 and found no evidence that the Rad 51-mediated increase is affected by the functional status of p53, a conclusion supported by the observed cytoplasmic localisation of p53 in ES cells following electroporation. Furthermore, in the absence of additional Rad51, p53-deficient ES cells do not have elevated rates of homologous recombination with extrachromosomal DNA. These findings demonstrate that Rad51 levels modify both homologous and illegitimate recombination, but that these phenomena are independent of p53 status

Effects of donor oocytes and culture conditions on development of cloned mice embryos

Mice have been successfully cloned from somatic and embryonic stem (ES) cells using the "Honolulu method." In the present study, different donor oocytes and different culture conditions were compared to evaluate the developmental potential of nuclear transfer embryos reconstructed with an inbred ES cell line HM-1. Oocytes were recovered from two different F1 donors B6D2F1 (C57BL/6 × DBA/2) and B6CBAF1 (C57BL/6 × CBA). There was no effect of oocyte origin on development of cloned embryos to the morulae/blastocyst stage (B6D2F1 44.1% vs. B6CBAF1 45.0%), and the transferred embryos could develop to term. Two culture conditions were compared to show their ability to support development to the morulae/blastocyst stage of reconstructed embryos with B6D2F1 oocytes. The total cell number in the cloned blastocysts cultured in M16 with 20% oxygen was much higher than that observed in CZB with 20% oxygen. Low oxygen concentration during culture of nuclear transfer embryos in CZB medium showed no beneficial effect on pre-implantation development, no embryos developed to term after transfer to surrogate mothers. Our results demonstrated that not only B6D2F1, but B6CBAF1 oocytes, can be used for nuclear transfer. M16 medium is superior for culture of nuclear transfer embryos and low oxygen concentration with CZB medium during culture shows no benefit on development of cloned embryos