Cytogenetic behavior of cryoprotectant DMSO

IVF (in vitro fertilization) is now used worldwide to overcome female or male infertility. Cryopreservation of human embryos provides the clearest opportunity to improve the clinical results obtained with IVF. Cryoprotective agents (CPA) are used to minimize freezing injuries. DMSO has been the most widely used CPA, however, high concentrations of CPAs in the vitrification solution have been shown to be detrimental to the cell. In order to determine the effect of DMSO solutions (5%, 10% and 20%) on genetic stability and/or subsequent DNA repair, we have investigated its ability to induce Sister Chromatid Exchanges (SCEs) and Proliferation Rate Index (PRI) in normal human lymphocyte cultures of peripheral blood, due to the fact that the study cannot be conducted on embryos and to the limited number of spare available embryos, the corresponding accessible experimental material was T lymphocyte. The blood samples were taken from three different healthy donors (conducting experimental procedure in triplicate). After the effect of DMSO solutions on blood according to the instructions of kit K-SIBV-500, lymphocytes are harvested and cultured with suitable technique to assess SCEs and PRI. The results show that all three DMSO concentrations cause a statistically dose depended significant increase of SCE frequency of the lymphocytes (p<0.001) and raise the need for more research regarding the safe and effective use of cryoprotectant

Aneuploidy in pluripotent stem cells and implications for cancerous transformation

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation

Is the sperm centrosome to blame for the complex polyploid chromosome patterns observed in cleavage stage embryos from an OAT patient?

Oligoasthenoteratozoospermia (OAT) is defined by a combined low count < 20 x 10(6) sperm/ml, poor motility < 50 % forward progression or < 25 % rapid linear progression and abnormal morphology (5-8% normal using Kruger strict criteria) and has been associated with increased levels of sperm aneuploidy. Here we report on the cytogenetic findings from three 'spare' embryos from a couple that were referred for ICSI because of OAT. The embryos were processed for sequential FISH in three hybridization rounds using probes for chromosomes 3, 7, 9, 13, 17, 18, 21, X and Y. Molecular cytogenetic analysis of nine chromosomes revealed that all three embryos were female polyploid. One of them was uniformly tetraploid for all chromosomes tested, while the remaining two embryos showed evidence of abnormal postzygotic segregation of chromosomes, causing the derivative blastomeres to have uneven chromosomal constitution. In one of them in particular, the non-disjoining chromosomes showed preferential segregation to the same pole, rather than randomly moving towards either pole, suggesting an abnormal spindle and causing the derivative blastomeres to have significantly uneven chromosomal constitutions. The possible scenarios leading to polyploidy and chromosomal imbalance through cytokinetic failure and subsequent abnormal centrosomal distribution are outlined

The effects of postnatal exposure of endocrine disruptors on testicular function: a systematic review and a meta-analysis

Background: Despite many epidemiological studies having been conducted, the impact of postnatal exposure of endocrine disruptors (EDs) on testicular function remains a controversial issue. Aim: To systematically review the literature and perform a quantitative synthesis to evaluate the effect of EDs on testicular function. Materials and methods: A comprehensive search was conducted in the MEDLINE, Scopus, and CENTRAL databases. Eligible for the systematic review were observational (cross-sectional and cohort) studies with (i) adult men who had a high probability of postnatal exposure to EDs (“exposed”), (ii) adult men who had a low probability of postnatal exposure to EDs (“non-exposed”), and (iii) an outcome of interest [seminal parameters and reproductive hormone concentrations]. The continuous outcomes in each of the studies were synthesized by the random effects model and expressed as standardized mean difference (SMD) with 95% confidence interval (CI). Results: Thirteen studies, including 959 exposed and 907 non-exposed men, fulfilled the inclusion criteria. Exposure to EDs was associated with decreased LH [SMD − 0.17, 95% CI − 0.33 to − 0.02, 10 studies (616 exposed, 563 non-exposed), I2 40%, p = 0.09], progressive motility [SMD − 0.45, 95% CI − 0.77 to − 0.13, three studies (133 cases, 153 controls), I2 38%, p = 0.20], and normal morphology [SMD − 0.50, 95% CI − 0.85 to − 0.14, eight studies (562 cases, 540 controls), I2 87%, p &amp;lt; 0.01] compared with non-exposure. No difference was observed between the other study groups. Conclusions: Postnatal exposure to EDs is associated with decreased semen quality. Nevertheless, there is no evidence that a disruption of testicular function mediates the deterioration in semen quality. © 2020, Hellenic Endocrine Society

The impact of sars-cov-2 on sperm cryostorage, theoretical or real risk?

Cryopreservation of human gametes and embryos as well as human reproductive tissues has been characterized as an essential process and aspect of assisted reproductive technology (ART). Notably, sperm cryopreservation is a fundamental aspect of cryopreservation in oncological patients or patients undergoing gonadotoxic treatment. Given that there is a risk of contamination or cross-contamination, either theoretical or real, during the procedures of cryopreservation and cryostorage, both the European Society for Human Reproduction and Embryology (ESHRE) and the American Society for Reproductive Medicine (ASRM) have provided updated guidelines for preventing or reducing the contamination risk of sexually transmitted viruses. Given the ongoing and worldwide COVID-19 pandemic, there is considerable interest in what measures should be taken to mitigate SARS-CoV-2 contamination during cryopreservation and cryostorage of semen samples. The SARS-CoV-2 virus is the virus that causes COVID-19, and whose transmission and infection is mainly aerosol-mediated. Several ART professional societies, including ESHRE and ASRM have proposed measures to mitigate the spread of the SARS-CoV-2 virus. Whether the proposed safety directives are enough to mitigate the possible SARS-CoV-2-contamination of sperm samples during cryopreservation or whether the policies should be re-evaluated will be discussed in this review. Additionally, insights regarding the possible impact of COVID-19 vaccination on the safety of sperm cryopreservation will be discussed. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Artificial oocyte activation: physiological, pathophysiological and ethical aspects

Infertile couples with low oocyte yield in combination with abnormal semen parameters may experience intra-cytoplasmic sperm injection (ICSI) failure. An established factor associated with ICSI failure is oocyte activation deficiency (AOD). The latter originates from seminal contributors, such as phospholipase C-zeta (PLCζ) that is not adequate to produce calcium (Ca 2+ ) oscillations for oocyte activation. Apart from this natural activator, other stimulants, such as A23187, ionomycin, strontium chloride or even electric pulses, have been used in embryological laboratories to overcome AOD and ICSI failure. The aim of the present narrative review is to discuss the role of Ca +2 oscillations in oocyte activation and summarize the evidence concerning the use of oocyte activators as agents for artificial oocyte activation (AOA). Studies in humans and animals have emerged many physiological, pathophysiological and ethical aspects of AOA. In conclusion, in mammalian eggs, the cytosolic Ca +2 oscillations derive from a periodic release of Ca +2 from intracellular pools. PLCζ, as well as artificial stimulants, have been used to produce Ca +2 oscillations for AOA. As the latter may increase the risk of epigenetic induced malformations, further studies are required to clarify whether AOA constitutes an effective and safe method to overcome ICSI failure. Abbreviations: AOA: artificial oocyte activation; AOD: oocyte activation deficiency; Ca +2 : Calcium; CAMKII: Ca +2 /calmodulin-dependent protein kinase II; CICR: calcium-induced calcium-release; DAG: diacylglycerol; GM-CSF: granulocyte-macrophage colony-stimulating factor; ICSI: intra-cytoplasmic sperm injection; InsP 3 R: inositol-trisphosphate receptor; IP 3 : inositol 1,4,5-trisphosphate; IVF: in vitro fertilization; MAP: mitogen-activated protein; MII: metaphase II; NADP: nicotinic acid adenine dinucleotide phosphate; NO: nitric oxide; PAWP: post-acrosomal WW-binding domain protein; PIP 2 : phosphatidylinositol 4,5-bisphosphate; PLC: phospholipase C; PLCζ: phospholipase C-zeta; SOAFs: spermatozoon-released oocyte-activating factors; Sr +2 : strontium; TFF: total fertilization failure. © 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group

Artificial oocyte activation: physiological, pathophysiological and ethical aspects

Infertile couples with low oocyte yield in combination with abnormal semen parameters may experience intra-cytoplasmic sperm injection (ICSI) failure. An established factor associated with ICSI failure is oocyte activation deficiency (AOD). The latter originates from seminal contributors, such as phospholipase C-zeta (PLCζ) that is not adequate to produce calcium (Ca 2+ ) oscillations for oocyte activation. Apart from this natural activator, other stimulants, such as A23187, ionomycin, strontium chloride or even electric pulses, have been used in embryological laboratories to overcome AOD and ICSI failure. The aim of the present narrative review is to discuss the role of Ca +2 oscillations in oocyte activation and summarize the evidence concerning the use of oocyte activators as agents for artificial oocyte activation (AOA). Studies in humans and animals have emerged many physiological, pathophysiological and ethical aspects of AOA. In conclusion, in mammalian eggs, the cytosolic Ca +2 oscillations derive from a periodic release of Ca +2 from intracellular pools. PLCζ, as well as artificial stimulants, have been used to produce Ca +2 oscillations for AOA. As the latter may increase the risk of epigenetic induced malformations, further studies are required to clarify whether AOA constitutes an effective and safe method to overcome ICSI failure. Abbreviations: AOA: artificial oocyte activation; AOD: oocyte activation deficiency; Ca +2 : Calcium; CAMKII: Ca +2 /calmodulin-dependent protein kinase II; CICR: calcium-induced calcium-release; DAG: diacylglycerol; GM-CSF: granulocyte-macrophage colony-stimulating factor; ICSI: intra-cytoplasmic sperm injection; InsP 3 R: inositol-trisphosphate receptor; IP 3 : inositol 1,4,5-trisphosphate; IVF: in vitro fertilization; MAP: mitogen-activated protein; MII: metaphase II; NADP: nicotinic acid adenine dinucleotide phosphate; NO: nitric oxide; PAWP: post-acrosomal WW-binding domain protein; PIP 2 : phosphatidylinositol 4,5-bisphosphate; PLC: phospholipase C; PLCζ: phospholipase C-zeta; SOAFs: spermatozoon-released oocyte-activating factors; Sr +2 : strontium; TFF: total fertilization failure. © 2018, © 2018 Informa UK Limited, trading as Taylor &amp; Francis Group

Insights into the role of telomeres in human embryological parameters. Opinions regarding ivf

Telomeres promote genome integrity by protecting chromosome ends from the activation of the DNA damage response and protecting chromosomes from the loss of coding sequences due to the end replication problem. Telomere length (TL) is progressively shortened as age progresses, thus resulting in cellular senescence. Therefore, TL is in strong adverse linear correlation with aging. Mounting evidence supports the notion that telomeres and male/female infertility are in a close relationship, posing the biology of telomeres as a hot topic in the era of human-assisted reproduction. Specifically, the length of sperm telomeres is gradually increasing as men get older, while the telomere length of the oocytes seems not to follow similar patterns with that of sperm. Nonetheless, the telomere length of the embryos during the cleavage stages seems to have a paternal origin, but the telomere length can be further extended by telomerase activity during the blastocyst stage. The latter has been proposed as a new molecular biomarker with strong predictive value regarding male infertility. As far as the role of telomeres in assisted reproduction, the data is limited but the length of telomeres in both gametes seems to be affected mainly by the cause of infertility rather than the assisted reproductive therapy (ART) procedure itself. The present review aims to shed more light into the role of telomeres in human embryological parameters, including gametes and embryos and also presents opinions regarding the association between telomeres and in vitro fertilization (IVF). © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Transdermal testosterone pretreatment in poor responders undergoing ICSI: A randomized clinical trial

STUDY QUESTION: Does pretreatment with transdermal testosterone increase the number of cumulus-oocyte complexes (COCs) retrieved by more than 1.5 in poor responders undergoing intracytoplasmic sperm injection (ICSI), using recombinant follicle stimulating hormone (FSH) and gonadotrophin releasing hormone agonists (GnRHa)? SUMMARY ANSWER: Testosterone pretreatment failed to increase the number of COCs by more than 1.5 as compared with no pretreatment in poor responders undergoing ICSI (difference between medians: 0.0, 95% CI: -1.0 to +1.0). WHAT IS KNOWN ALREADY: Androgens are thought to play an important role in early follicular development by enhancing ovarian sensitivity to FSH. In a recent meta-analysis, testosterone pretreatment resulted in an increase of 1.5 COCs as compared with no pretreatment. However, this effect was based on the analysis of only two randomized controlled trials (RCTs) including 163 patients. Evidently, there is a need for additional RCTs that will allow firmer conclusions to be drawn. STUDY DESIGN, SIZE, DURATION: The present RCT was designed to detect a difference of 1.5 COCs (sample size required = 48 patients). From 02/2014 until 04/2015, 50 poor responders fulfilling the Bologna criteria have been randomized (using a randomization list) to either testosterone pretreatment for 21 days (n = 26) or no pretreatment (n = 24). PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients underwent a long follicular GnRHa protocol. Recombinant FSH stimulation was started on Day 22 following GnRHa initiation. In the testosterone pretreatment group, a daily dose of 10 mg of testosterone gel was applied transdermally for 21 days starting from GnRHa initiation. Results are expressed as median (interquartile range). MAIN RESULTS AND THE ROLE OF CHANCE: No differences in baseline characteristics were observed between the two groups compared. Testosterone levels [median (interquartile range)] were significantly higher in the testosterone pretreatment on the day of initiation of FSH stimulation [114 (99.5) ng/dl versus 20 (20) ng/dl, respectively, P < 0.001]. Duration of FSH stimulation [median (interquartile range)] was similar between the groups compared [12.5 (3.0) days versus 12 (3.0) days, respectively, P = 0.52]. The number of COCs retrieved [median (interquartile range)] was not different between the testosterone pretreatment and the no pretreatment groups [3.5 (4.0) versus 3.0 (3.0), 95% CI for the median: 2.0-5.0 versus 2.7-4.3, respectively; difference between medians: 0.0, 95% CI: +1.0 to -1.0). Similarly no differences were observed regarding fertilization rates [median (interquartile range)] [66.7% (32.5) versus 66.7% (42.9), respectively, P = 0.97] and live birth rates per randomized patient (7.7% versus 8.3%, respectively, rate difference: -0.6%, 95% CI: -19.0 to +16.9). LIMITATIONS, REASONS FOR CAUTION: The study was not powered to detect differences less than 1.5 COCs, although it is doubtful whether these differences would be clinically relevant. Moreover, due to sample size restrictions, no conclusions can be drawn regarding the probability of live birth. WIDER IMPLICATIONS OF THE FINDINGS: The results of this randomized clinical trial, suggesting that pretreatment with 10 mg of transdermal testosterone for 21 days does not improve ovarian response by more than 1.5 oocytes, could be used to more accurately consult patients with poor ovarian response. However, an improvement in IVF outcome using a higher dose of testosterone or a longer pretreatment period cannot be excluded. STUDY FUNDING/COMPETING INTEREST: The study was partially funded by a Scholarship from the Academy of Athens. C.A.V. reports personal fees and non-financial support from Merck, Sharp and Dome, personal fees and non-financial support from Merck Serono, personal fees and non-financial support from IPSEN Hellas S.A., outside the submitted work. B.C.T. reports grants from Merck Serono, grants from Merck Sharp & Dohme, personal fees from Merck Serono, personal fees from Merck Sharp & Dohme, personal fees from IBSA & Ferring, outside the submitted work. TRIAL REGISTRATION NUMBER: NCT01961336. TRIAL REGISTRATION DATE: 10 October 2013. DATE OF FIRST PATIENT'S ENROLLMENT: 02/2014. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved