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Failure of complete hatching of ICSI-derived human blastocyst by cell herniation via small slit and insufficient expansion despite ongoing cell prolif
ICSI-derived blastocysts intermittently release proliferating cells and extracted TE cells and/or inner cell masses via a small slit: thus, blastocyst expansion is not sufficiently increased, leading to a reduced complete hatching rate. Therefore, the ICSI penetration trace potentially has negative effects on blastocyst expansion process in vitro and is a risk factor for the failure of completed hatching.
The composition of human preimplantation embryo culture media and their stability during storage and culture
The presence of D-lactate could be avoided and the finding of human liver enzymes was surprising. The wide variation between culture media shows that the optimal composition is still unknown. This warrants further research as the importance of embryo culture media on the efficacy and safety in IVF is evident. Companies are urged to fully disclose the composition of their culture media, and provide clinical evidence supporting the composition or f
Novel phospholipase C zeta 1 mutations associated with fertilization failures after ICSI
PLCZ1 mutations were found in high frequency in patients presenting OAF. Functional analysis of three mutations in human oocytes confirms alteration of PLCζ::/PLCZ1 activity and their likely involvement in impaired oocyte activation. Our results suggest that PLCZ1 gene sequencing could be useful as a tool for the diagnosis and counseling of couples presenting FF after ICSI due to OAF.
Transgenerational inheritance: how impacts to the epigenetic and genetic information of parents affect offspring health
The environment influences the health and well-being of progeny by working through the germline to introduce spontaneous genetic mutations as well as a variety of epigenetic changes, including alterations in DNA methylation status and the post-translational modification of histones. In evolutionary terms, these changes create the phenotypic diversity that fuels the fires of natural selection. However, rather than being adaptive, such variation ma
Female obesity is negatively associated with live birth rate following IVF: a systematic review and meta-analysis
WIDER IMPLICATIONS: Our meta-analysis clearly demonstrates that female obesity negatively and significantly impacts live birth rates following IVF. Whether weight loss can reverse this deleterious effect through lifestyle modifications or bariatric surgery should be further evaluated.
The enigmatic morula: mechanisms of development, cell fate determination, self-correction and implications for ART
In clinical embryology, the morula stage has been always perceived as a ‘black box’ in the continuum of preimplantation development. This has dictated its virtual exclusion from mainstream ART procedures. Recent findings described in this review indicate that the morula, and the associated process of compaction, as a crucial stage not only for the formation of the blastocyst, but also for the health of the conceptus. This understanding may open n
Activation of Toll-like receptor 7/8 encoded by the X chromosome alters sperm motility and provides a novel simple technology for sexing sperm
Ligand activation of TLR7/8 selectively suppressed the mobility of the X chromosome–bearing sperm (X-sperm) but not the Y-sperm without altering sperm viability or acrosome formation. The difference in sperm motility allowed for the separation of Y-sperm from X-sperm. Following in vitro fertilization using the ligand-selected high-mobility sperm, 90% of the embryos were XY male. Likewise, 83% of the pups obtained following embryo transfer were XY
Environmental Exposure of Sperm Sex-Chromosomes: A Gender Selection Technique
The results indicated successful enrichment of Xchromosome-bearing spermatozoa upon incubation in acidic media, increased temperatures, and elevated H2O2. This study demonstrated the potential role for exploring the physiological differences between X-and Y-chromosome-bearing spermatozoa in the development of preconceptual gender selection.
A simpler way to choose the sex of offspring by separating X and Y sperm.Differential gene activity by the two sex chromosomes allows X-bearing sperm
A simple, reversible chemical treatment can segregate X-bearing sperm from Y-bearing sperm, allowing dramatic alteration of the normal 50/50 male/female offspring ratio, according to a new study. The study was performed in mice, but the technique is likely to be widely applicable to other mammals as well.
The human placenta may not have a microbiome after all.
Read more: https://www.newscientist.com/article/2211529-the-human-placenta-may-not-have-a-m
Is the placenta home to communities of bacteria? Recent evidence that the organ has its own microbiome promised to rewrite our understanding of it, but now research suggests that the earlier experiments may have been contaminated.
In some components, all culture media are similar but in others they vary widely. Moreover, the concentrations of some components will vary with storage and culture.
Comment
It’s funny that while one of the key elements in the continued improvement in IVF success rates have been in the development of the embryo culture medium, the actual concentration of each listed component is confidential. All IVF clinics have largely signed over their operations to media manufacturers at considerable costs to what are now large companies. Many embryologists will probably be unsure of the role of each component and therefore in a poor position to make an informed decision about which media to employ. Yet the health of each child surely has some dependency of its environment prior to implantation. |In a subscription-based article published in Human Reproduction, the authors from a Dutch laboratory have examined the composition of 15 culture medias from 7 manufactures covering most variations now available including sequential and continuous formulations. They reported both on their initial analysis and the changes observed after storage and mock embryo culture. Their reports make for some reassuring and some disturbing reading.|Given the media may be either sequential or continuous, it is not surprising the media formulations varied both in their ionic and energy substrates or amino acids concentrations. However, it may be fair to expect that there should be an optimal formulation given they are all used on the same human embryos. Perhaps in 2019, the industry should expect all embryos to be uniformly cultured. How can epidemiologists compare developmental outcomes of children if there remains significant variation in culture environments? Data collection agencies do not collect the type of culture media employed and until it is, there remains a weak link in the accountability of IVF. Perhaps the manufacturers should compete on quality and service rather than on media formulation.| The authors did report the presence of D-latacte in two media which was unexpected since d-lactate cannot be used by the embryo and may possible be detrimental? The osmolarity also varied between the sequential and complete medias between 270 and 299 mOsmol. The bottom line is that even though most media will report similR constituent profiles, the actual concentrations may vary and each clinic should at least to establish the what and why of each component of their media. Also, if a clinic chooses to mix media say between cleavage and blastocyst stages then they should check of osmolarity differences to avoid metabolically stressing the embryo.|The authors also reported on what happens to the media when it was stored and sham cultured and the changes they reported, while small, were real. The changes, especially in the concentration of amino acids were unpredictable and concerning. They explored if this as due to absorption by the mineral oil overlay and found the oil unchanged. They then tested the protein source for contamination and reported the presence of enzymic activity in some albumin preparations that may explain the changes. The authors only tested for the presence of two enzyme so possibly, there may be further contamination. These observations need to be expanded and repeated but the suggestion that the media is not stable is of interest. As part of a clinics quality management system could include the time media is out of cold storage and place limits on their exposure to room temperatures. Single use media may also be another way manufacturers can guaranteed the quality of their media.|Most scientists are aware that the preparation and testing of media is both extensive and expensive and no longer within their capabilities. Changing media formulations require multiple regulative hurdles to be meet so changing them is challenging. This article does reinforce the clinics responsibility to ask questions of their media manufacturer about their formulations and quality control.|
Title
Can the sex of individual spermatozoa be determined by their motility ?
Summary
In this study from Japan, the authors have used exciting new gender specific ligand binding to separate X and Y bearing spermatozoa.
Comment
In mammals, sex is determined by the presence of the X chromosome or Y chromosome. Male spermatogonial stem cells proliferate by mitosis and then enter meiosis where each spermatid acquires either the X or Y chromosome. These haploid germ cells differentiate to spermatozoa and after fertilization the resulting zygotes have either two X chromosomes (XX female zygotes) or an X and Y chromosome (XY male zygotes). X-chromosome has more than 1,000 kinds of functional genes and the transcription is active in haploid male germ cell. |However, it is generally assumed that the X and Y haploid spermatogonia are functionally similar. Using the cytoplasmic bridges between spermatids, cytoplasm including RNAs and proteins are shared to rescue Y chromosome bearing sperm (Y-sperm). It has been reported that the bridge works in spermatid at early stage of spermiogenesis and the high levels of RNA polymerase II are detected in this stage. Therefore, both types of sperm carrying X-chromosome and sperm carrying Y-chromosome have same fertilization ability, which results in 1:1 sex ratio of male to female offspring in mammales. |However, there is also evidence that the X- and Y-sperm respond differentially to external signals, such as pH. We hypothesized that functional differences between X-sperm and Y-sperm might be related to the expression of specific genes that were expressed in haploid male germ cells after the bridges were lost, especially those encoding receptors that respond to signals from the external environment. We show herein for the first time that Toll-like receptor 7 (TLR7) and TLR8 are expressed exclusively by the X chromosome in the haploid male germ cells, that ligand activation of TLR7 and TLR8 alters X-sperm functions and mobility and that by selectively changing X-sperm mobility, X- and Y-sperm can be easily and selectively isolated by a novel technique. Thus, not only do we show that TLR7 is functional in X-sperm but not Y-sperm but this study offers a technique for sexing sperm.|
Specifically, in this study, RNA sequence analyses were done on mouse spermatozoa and revealed that the X chromosome encoded 492 genes, including TLR7 and TLR8. TLR7 and TLR8 were expressed in half of the testicular round spermatids. All of TLR7/8 positive cells were stained by anti-Sp56 antibody, a marker of acrosome, suggesting that these receptors were expressed in spermatids at the latter stage of spermiogenesis. Although we did not observe the proteins forming the cytosolic bridges between round spermatids, such as TEX14, it is one possibility that X-chromosome encoding proteins including TLR7/8 that are expressed at the latter stage of spermiogenesis would not be shared between X-spermatids and Y-spermatids. |In mature sperm, TLR7 was localized to the flagella and TLR8 was in midpiece of half of the spermatozoa. Moreover, the agonists of TLR7 and TLR8 suppressed ATP production by the glycolytic pathway or mitochondrial activity, resulting in decreased motility but not viability in the X-chromosome bearing sperm. Y-sperm that do not express TLR7 and TLR8 maintained high motility when sperm were treated with the agonists.
|Based on these observations, we utilized ligand activation of TLR7/8 to provide a convenient, effective, fast and simple method for the selectively separating Y-sperm from X-sperm by the reducing the velocity of X-sperm. Specifically, the incubation of sperm for 1 hr with TLR7/8 ligands efficiently separated X-sperm from Y-sperm without decreasing fertilization, at least in vitro. Gledhill et al. (1983) focused on the length of the sex chromosome and developed the separation method of X-sperm and Y-sperm by cell sorting of sperm stained with Hoechst 33342. However, this method based on flow cytometry and cell sorting has several limitations. The ultraviolet rays used to detect fluorescence in sperm by flow cytometry decreases sperm fertilization capacity. Furthermore, flow cytometry with a cell sorting system is too expensive to introduce locally at each farm. Because the expression of the Tlr7/8 gene is highly conserved on the mammalian X chromosome including cattle, pig and human, our novel protocol using TLR7/8 ligand activation has high promise of being adapted to mammalian reproductive technologies for separating the X-sperm and Y-sperm within a short time and without costly, injurious and time-consuming cell sorting systems. |We have already adapted this method to cattle and pig production. Using in vitro fertilization technique in cattle, using sperm collected from upper layer of semen after the treatment with TLR7 and TLR8 agonists, XY embryos were generated more than 90% in cattle. From lower layer of semen treated with the agonists, more than 90 % of embryos were XX female embryo.|
We know that TLR7 and TLR8 are encoded by X-chromosome in human. Although we did not try to detect the expression of them in human sperm, we think that the method is available for human in vitro fertilization. However, use of this method in human reproductive technology is speculative at the moment, and involves significant ethical issues unaffected by the utility of this new technique.|
IVFCPD thanks Dr Shimada for this summary of his work.
Title
Can separating X and Y bearing spermatozoa be this easy?
Summary
In a PLOS Biology paper, it appears (in mice) that understanding what receptors are coded by the X-chromosome may reveal an easy way for X and Y spermatozoa to be separated in a non-toxic manner.
Comment
In an open access paper in PLOS Biology, a group of Japanese workers have published information that implies the gender ratio of embryos after (mouse) IVF can be manipulated about 90% into either females or males. The authors examined the genes located on the X-Chromosome of which there are about 3000, compared to about 700 on the Y chromosome. RNA from such gene activity is distributed via cytoplasmic bridges between spermatids some of which are critical for survival. However, some genes code for membrane receptors that are involved in metabolic activity and are only expressed on the membranes of X-bearing spermatozoa. The group the authors explored was Toll-like receptors. Wikipedia describes “Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single, membrane-spanning, non-catalytic receptors usually expressed on sentinel cells such as macrophages and dendritic cells, that recognize structurally conserved molecules derived from microbes". |Why they are expressed on spermatozoa remains unclear but very interesting. What was exciting was when these receptors were activated by agonists, sperm motility decreased in a reversible manner when the agonist was removed. The authors showed TL7 was expressed in the flagellum of the sperm and TL8 on the midpiece membrane. They then determined TL7 activation inhibits glycolysis by down regulating hexokinase and TL8 activation inhibited the TCA cycle by mitochondrial suppression. |They then exposed mouse sperm to TL7/8 agonists and the spermatozoa that were able to swim up a chamber were mostly Y-bearing spermatozoa while X-bearing spermatozoa, whose motility was significantly depressed remained largely in the lower part of the chamber. When sperm from the upper (male) and lower (female) chamber section were washed clean of the agonist and then used in IVF, low and behold, the gender ratio if blastocysts were skewed by about 90%.|The concept of this paper provides for an uncomplicated way to separated sperm on their functionality rather than other processes that are more invasive such as using dyes to estimate the amount of DNA in a sperm head. It does not have the efficiency of PGD but has the potential to be used in AIH. Even if used in IVF with PGD to confirm the gender of the embryos, there will be less embryo wastage if most of the embryos are of the required gender. The authors stated that TL7/8 are expressed in human spermatozoa and it appears some of the agonist used are also used in human clinical interventions so it will be interesting to see if future further work reinforces their observations and whether passive gender separation of sperm may be acceptable in clinical IVF to allow couples some control (but not guaranteed) success in having a child of the gender of their choice. |However, for the more academically minded, the real question is why does the receptors exist in the first place. If Toll-like receptors are activated by RNA from viral exposure as the authors noted, then does this suggest a role in changing gender outcomes in relation to infections? This may make sense if motility was suppressed in both X and Y bearing spermatozoa but why there may be a gender bias is intriguing. |
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