Preimplantation genetic testing (PGT) is the earliest form of prenatal diagnosis that has become an established procedure for couples at risk of passing a severe genetic disease to their offspring. ...At UMC Ljubljana, we conducted a retrospective register-based study to present 15 years of PGT service within the public healthcare system in Slovenia. We collected the data of the PGT cycles from 2004 to 2019 and compared clinical outcomes for chromosomal and monogenic diseases using different embryo biopsy and testing approaches. In addition, we assessed the extent to which PGT has become the preferred option compared to classic prenatal diagnostics. We treated 211 couples, 110 with single gene disorder, 88 with structural chromosome rearrangement and 13 for numerical chromosome aberration. There were 375 PGT cycles with oocyte retrieval, while embryo transfer was possible in 263 cases resulting in 78 deliveries and 84 children. Altogether, the clinical pregnancy rate per embryo transfer was 31% in 2004–2016 (blastomere biopsy) and 43% in 2017–19 (blastocyst biopsy), respectively. We assessed that approximately a third of couples would opt for PGT, while the rest preferred natural conception with prenatal diagnosis. Our results show that providing a PGT service within the public healthcare system has become a considerable option in pregnancy planning for couples at risk of transmitting a severe genetic disease to their offspring. In Slovenia, approximately a third of couples would opt for PGT. Although the number of cycles is small, our clinical results are comparable to larger centres.
Aspirated follicular cells (AFCs) from the in vitro fertilization program can express various stem cell markers and are even able to differentiate into different types of cells in vitro. The female ...reproductive potential decreases with increasing age due to lowered ovarian reserve and oocyte quality, but data on the effect of female age on stem cell characteristics of AFCs are scarce. Therefore, the aim of this study was to elucidate whether female age affects the mesenchymal stem cell (MSC) characteristics of AFCs. Follicular aspirates were collected from 12 patients included in the in vitro fertilization programme with a normal ovarian reserve. Patients were divided into four age groups: Group A ≤ 30 years, Group B 31–35 years, Group C 36–39 years and Group D ≥ 40 years. After removal of the oocytes, AFCs were collected from follicular aspirates using hypo-osmotic technique and cultured in vitro, and their stemness was compared according to female age. The cultured AFCs were analysed for gene expression using the Human Mesenchymal Stem Cell RT
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Profiler™ PCR Array, for their potential for differentiation into adipogenic and osteogenic lineage, and for their expression of MSC-related markers using immunocytochemistry. We found that female age can significantly influence their stemness: expression of pluripotency and MSC-related genes, and their differentiation potential. Despite the relatively high expression of MSC-related genes, the AFCs of the oldest patients had the lowest potential to differentiate into osteogenic and adipogenic lineages in vitro, which may be related to their age and the changed ovarian function.
Abstract
Study question
Does vitrification provide a better method for human sperm cryopreservation than slow freezing in terms of survival, mobility, maturity, and DNA fragmentation?
Summary answer
...Vitrification of human semen yields similar outcome as slow-freezing considering survival, mobility, DNA fragmentation, and maturity but the mobility after thawing is lower.
What is known already
Slow freezing is the most common technique for cryopreservation of human sperm. Unfortunately, many sperm cells die during the process or lose their mobility and are thereafter unable of fertilization. Meanwhile, vitrification of animal sperm showed good outcomes in terms of sperm survival and motility while being easier to perform than slow freezing. So far, many studies focused on survival and mobility of human sperm after vitrification compared to slow freezing, with results still not unified. They also neglected the importance of sperm maturity and DNA fragmentation caused by the process. DNA damage can affect embryo development or cause abortion.
Study design, size, duration
The study was performed from March January to July 2022. It included 69 male participants who underwent a diagnostic spermiogram at the Division of gynaecology and obstetrics of University Medical Centre Ljubljana. All participants gave written consent of using the remaining of sample, after performing the spermiogram, for our study. Sperm samples were divided in three aliquots, one saved as native sample, one intended for vitrification, and one intended for slow freezing.
Participants/materials, setting, methods
Participants in the study had normal sperm quality parameters according to 5th edition of WHO manual. Slow freezing was performed using Sperm Freezing medium (Origio). For vitrification sperm was diluted in solution consisting of Sperm preparation medium (Origio), sucrose, and HSA in 1:1 (v/v) ratio, incubated for five minutes and then vitrified by adding 30 microliter droplets directly into liquid nitrogen. After thawing sperm survival, mobility, maturity, and DNA fragmentation were assessed.
Main results and the role of chance
The data are presented as mean values (± standard deviation) when normally distributed and as medians (IQR) when not normally distributed. The patient’s age was 35.0±5.1 years. In native sperm sample, sperm concentration was 69.6±59.0x106 cells per millilitre and total motility was assessed to be 60.0% (50.0%-70.0%). After thawing we noticed significantly lower mobility (p < 0.001), while also the mobility was significantly lower after vitrification when compared to slow-freezing (17.6% (12.4%-25.3%) and 14.5% (9.0%-22.6%), p = 0.007). Interestingly, there was no difference in survival after thawing the samples (20.5±10.7 % after slow freezing and 23.7±13.7 % after vitrification). Also there was no difference in maturity of sperm when checked with protamine staining (native vs. slow-freezing vs. vitrification; 53.6±15.2 % vs. 53.9±16.0 % vs. 52.4±15.2 %, p = 0.905) and also when checked with hyaluronan binding assay (78.0% (67.5%-82.4%) vs. 79.4% (71.7%-82.95%) vs. 75.3% (67.5%-81.1%), p = 0.607) . While sperm maturity was not affected with cryopreservation, DNA fragmentation was significantly higher after cryopreservation (native vs. slow-freezing vs. vitrification; 19.7±9.5%, vs. 24.7±11.6 vs. 27.1±15. %, p = 0.049).
Limitations, reasons for caution
We believe our study would be better by obtaining higher number of participants and by being performed over longer time period. We would also like to further optimise vitrification protocol. More knowledge is also needed in poor sperm samples, since we only included sperm samples of good quality.
Wider implications of the findings
We believe our finding are of great importance for the field of andrology. Our results show vitrification of human sperm can be comparable to slow freezing in most of parameters analysed, but lower mobility after vitrification is reason for caution.
Trial registration number
20210024
Abstract
Study question
How does significant increase of the proportion of single embryo transfer over 10 years period affect pregnancy, live birth and twin rate.
Summary answer
Increase in single ...embryo transfer doesn’t change pregnancy and live birth rate, although it significantly lowers twin rate.
What is known already
Due to widely used approach in IVF of transferring multiple embryos to improve the pregnancy and birth rate, multiple pregnancies, mostly twin, are quite common. But because they are more often associated with adverse neonatal and perinatal outcomes as singleton pregnancies, they are not desirable. Therefore, more and more often the transfer of single embryo is encouraged. Furthermore, in the last years with improvements in cryopreservation techniques leading to effective cryopreservation of supernumerary embryos, there is more options for performing repeated single embryo transfers.
Study design, size, duration
We retrospectively collected the data of all fresh embryo transfers in couples treated in our centre from January 2010 to December 2019. We excluded embryo transfer where embryos were derived from cryopreserved oocytes and analysed the outcome of fresh embryo transfer regarding to the number of transferred embryos.
Participants/materials, setting, methods
In our analysis we included 10583 fresh embryo transfers. We tried to evaluate how the proportion of single embryo transfer has changed through analysed period of time and if this led to any differences in pregnancy, live birth, and twin rate. To determine the differences between the groups, the data were analysed with one-way ANOVA and Pearson’s chi-square, as appropriate. Statistical significance was set at P < 0.05.
Main results and the role of chance
The analysis revealed that the proportion of single embryo transfers significantly increased from year 2010 to year 2019 (from 28% to 73%; P < 0.001). The proportion increased every year, minimum increase was 1% whereas maximum increase was 16%. This increase over the years did not negatively affect the pregnancy (32% in 2010 vs. 34% in 2019: p = 0.317) and live birth rates (24% vs. 25%; p = 0.584), although it had favorable effect on twin rate (16% vs. 7%; p = 0.002). If we separately analyzed only single and double embryo transfer, we observed that pregnancy (24% in 2010 and 34% in 2019; p = 0.001) and live birth rates (17% vs. 26%, p = 0.001) significantly increased after single embryo transfers, but no difference was observed in double embryo transfers (pregnancy rate: 35% vs. 35%, p = 1; live birth rate: 27% vs. 22%, p = 0.097; twins rate: 20% vs. 27%, p = 0.244). Additionally, we observed that female mean age value significantly increased over analyzed period (34.2±4.5 years in 2010 vs. 35.7±4.7 years in 2019, p < 0.001), although there was no difference in mean number of retrieved oocytes (8.2±5.4 vs. 8.1±4.9, p = 1) and obtained embryos (4.5±3.3 vs. 4.2±2.9; p = 0.684).
Limitations, reasons for caution
The limitation of the study is retrospective design, and not evaluating the influence of elective single embryo transfer. Also, the IVF laboratory methods and IVF culture media improved over the years meaning they could be partly responsible for observed differences.
Wider implications of the findings: Single embryo transfer could probably be performed in even higher proportion without lowering the chances for pregnancy.
Trial registration number
not applicable’
Background
DNA methylation patterns can show transgenerational inheritance and are influenced by lifestyle and environmental factors. It is suggested that these patterns can be changed by assisted ...reproductive technology.
Objectives
To evaluate the impact of two different sperm preparation methods, conventional density gradient centrifugation (DGC) vs. density gradient centrifugation followed by magnetic‐activated cell sorting (MACS) of non‐apoptotic spermatozoa, on sperm DNA methylation profile.
Materials and methods
We analyzed semen of patients included in our IVF treatment program. Half of the semen from each included patient was prepared for ICSI using the DGC method and the other half with DGC followed by MACS. The remaining samples were processed for DNA methylation analysis with reduced representation bisulfite sequencing (RRBS). In addition to the DNA methylation profile, we assessed the morphology and DNA fragmentation of spermatozoa.
Results
RRBS analysis revealed that the average genome‐wide methylation level was similar between both groups (DGC vs. MACS group) and ranged from 0.53 to 0.56. Furthermore, RRBS analysis identified 99 differentially methylated regions (DMRs) and 800 differentially methylated positions (DMPs). In the DGC group, 43 DMRs and 392 DMPs were hypermethylated whereas 56 DMRs and 408 DMPs were hypomethylated compared with those in the MACS group. When DMRs and DMPs were annotated to genes, 3 genes associated with imprinting were found: IGF2, PRDM16, and CLF4/BRUNOL4. The percentage of morphologically normal spermatozoa (MACS vs. DGC; 14.0 ± 10.8 vs. 13.2 ± 10.0; P = .335) and of spermatozoa with fragmented DNA of patients with RRBS analysis (22.9 ± 21.1% vs. 34.4 ± 21.2; P = .529) were also similar between groups.
Discussion and Conclusion
Although the average genome‐wide level of sperm DNA methylation was similar in both sample groups, a distinctive number of methylation changes were observed in DMR and DMP levels. A larger number of samples should be analyzed and additional sperm preparation methods should be tested to confirm our findings.
This review provides information to scientists performing animal surgery with recommendations for choosing an appropriate anesthetic agent. The human health risks from exposure to various anesthetic ...agents as well as methods to minimize exposure are discussed. In brief, methoxyflurane, used with precautions, is recommended for rodent bench-top surgery. When a precision vaporizer is available, isoflurane is usually the agent of choice. Other agents, including injectables, are considered.
In the present study, we investigated the radiosensitivity profiles of three established human ovarian carcinoma cell lines, PA-1, Caov-3, and SK-OV-3, using the adenosine triphosphate-cell viability ...assay (ATP-CVA). We have correlated radioresponsiveness with the p53 status and the p53 accumulation after irradiation as well as with the Bcl-2 expression and the growth rate of these cell lines. The p53 status was examined by immunocytochemistry and a functional assay (functional analysis of separated alleles in yeast, FASAY); the p53 accumulation was determined by immunocyotochemistry and flow cytometry. Futhermore, the Bcl-2 expression before and after irradiation was examined by immunocytochemistry. PA-1, expressing wild-type p53, showed an unequivocal accumulation of p53 protein following exposure to irradiation. This cell line was found to be strongly sensitive to irradiation. The two
p53 mutant cell lines Caov-3 and SK-OV-3 showed radioresistance at different degrees and irradiation did not result in p53 accumulation. None of the cell lines examined expressed Bcl-2 protein and no change was seen after irradiation. Furthermore, the most sensitive cell line to irradiation, PA-1, showed the highest proliferative activity, while Caov-3 and SK-OV-3, the more resistant cell lines, exhibited lower growth rates. Our findings indicate that the presence of p53 protein is a possible determinant for the cytotoxicity induced by irradiation in the investigated ovarian carcinoma cell lines. Bcl-2 expression does not seem to determine the response to irradiation in these cell lines. Additionally, an association between radioresponsiveness and the growth rate is suggested in PA-1, Caov-3, and SK-OV-3.
Class II Biological Safety Cabinets (BSCs) are widely used in biological and chemical research for protection of the investigator, the environment, and the project. However, researchers' operational ...procedures are often inconsistent with BSC design limitations. While the Class II BSC is only a partial containment cabinet, better design and user training can improve containment, thereby increasing personnel protection. The authors developed design modifications for a Class II BSC to permit internal waste collection and to optimize the available work area. An internal recessed well for the waste receptacle, relocation of petcocks and electrical duplex, and installation of a new vacuum bottle make operation of the cabinet more efficient and potentially safer. To correct poor work practices, which can compromise the protective features of any BSC, precise guidelines and training programs should be relied on. Standardization of guidelines for operation of BSCs would be beneficial to the clinical and research community, given the frequent exchange of investigators among biomedical institutions.
Extended work shifts of twelve hours or longer are common and even popular with hospital staff nurses, but little is known about how such extended hours affect the care that patients receive or the ...well-being of nurses. Survey data from nurses in four states showed that more than 80 percent of the nurses were satisfied with scheduling practices at their hospital. However, as the proportion of hospital nurses working shifts of more than thirteen hours increased, patients' dissatisfaction with care increased. Furthermore, nurses working shifts of ten hours or longer were up to two and a half times more likely than nurses working shorter shifts to experience burnout and job dissatisfaction and to intend to leave the job. Extended shifts undermine nurses' well-being, may result in expensive job turnover, and can negatively affect patient care. Policies regulating work hours for nurses, similar to those set for resident physicians, may be warranted. Nursing leaders should also encourage workplace cultures that respect nurses' days off and vacation time, promote nurses' prompt departure at the end of a shift, and allow nurses to refuse to work overtime without retribution.
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