Days Of Cryopreservation Research Articles

Association of duration of embryo culture with risk of large for gestational age delivery in cryopreserved embryo transfer cycles

To examine the relationship between the day of embryo cryopreservation and large for gestational age (LGA) infants in women undergoing frozen embryo transfers (FETs) after cryopreservation on days 2-7 after fertilization and to compare the risk of the day of embryo cryopreservation to other possible risk factors of LGA after FET cycles. Retrospective cohort study. Society of Assisted Reproduction Clinical Outcomes Reporting System. Women undergoing FET cycles. Day of cryopreservation. Singleton LGA infant. A total of 33,030 (18.2%) FET cycles in the study group (n = 181,592) resulted in LGA infants during the study period of 2014-2019. There was an increase in LGA risk when cryopreservation was performed from day 2 (13.7%) to days 3-7 (14.4%, 15.0%, 18.2%, 18.5%, and 18.9%). In the log-binomial model, the risk increased compared with days 2-3 combined when cryopreservation was performed on days 5-7 (adjusted relative risk [aRR] 1.32, 95% confidence interval [CI] 1.22-1.44 for day 5, aRR 1.34, 95% CI 1.23-1.46 for day 6, and aRR 1.42, 95% CI 1.25-1.61 for day 7). Other factors most associated with LGA risk in the log-binomial model were preterm parity of >3 compared with 0 (aRR 1.82, 95% CI 1.24-2.69) and body mass index (BMI) of >35 kg/m2 compared with normal weight (aRR 1.94, 95% CI 1.88-2.01). Increasing gravity, parity, BMI, number of oocytes, and embryo grade were also associated with LGA in this model. Asian, Black, Hispanic, and combined Hawaiian and Pacific Islander were protective factors in the model compared with White patients. Low BMI (<18.5 kg/m2) was also considered a protective factor in the model compared with normal BMI. Duration of embryo culture was associated with an increased risk of LGA in this study cohort when controlling for known confounders such as maternal BMI and parity. This study sheds new light on the possible link between FET and LGA infants.

Development of a Me2SO-free cryopreservation medium and its long-term cryoprotection on the CAR-NK cells

Cryopreservation is a crucial step in the supply process of off-the-shelf chimeric antigen receptor engineered natural killer (CAR-NK) cell products. Concerns have been raised over the clinical application of dimethyl sulfoxide (Me2SO) due to the potential for adverse reactions following infusion and limited cell-specific cytotoxic effects if misapplied. In this study, we developed a Me2SO-free cryopreservation medium specifically tailored for CAR-NK cells to address this limitation. The cryopreservation medium was formulated using human serum albumin (HSA) and glycerol as the base components. Following initial screening of seven clinically-compatible solutions, four with cryoprotective properties were identified. These were combined and optimized into a single formulation: IF-M. The viability, phenotype, and function of CAR-NK cells were evaluated after short-term and long-term cryopreservation to assess the effectiveness of IF-M, with Me2SO serving as the control group. The viability and recovery of CAR-NK cells in the IF-M group were significantly higher than those in the Me2SO group within 90 days of cryopreservation. Moreover, after 1 year of cryopreservation the cytotoxic capacity of CAR-NK cells cryopreserved with IF-M was comparable to that of fresh CAR-NK cells and significantly superior to that of CAR-NK cells cryopreserved in Me2SO. The CD107a expression intensity of CAR-NK cells in IF-M group was significantly higher than that of Me2SO group. No statistical differences were observed in other indicators under different cryopreservation times. These results underscore the robustness of IF-M as a suitable replacement for traditional Me2SO-based cryopreservation medium for the long-term cryopreservation and clinical application of off-the-shelf CAR-NK cells.

Stress Factors Affecting the Cryopreservation of Biological Components of Seed Virus for Avian Influenza Vaccine Production

In the past, many studies have been done to cryopreserve biological materials for future vaccine production. Scientists have been using different chemicals as cryoprotectants to preserve their cell lines on which desired viruses can be cultivated. Researchers have always been in search for better molecules to avoid cryoinjury during process of cryopreservation. In the present study, different molecules were evaluated for cryo-potency in preserving master seeds of &amp;ldquo;Vero cell&amp;rdquo; line and &amp;ldquo;Avian Influenza viruses&amp;rdquo;. Cryoprotectants such as (Dimethyl sulfoxide) DMSO and Glycerol were used in different concentration and evaluated at -80oC and -196oC for different time interval. After 15 days and 30 days of cryopreservation the percentage viability of preserved cells were almost equal at both temperatures whereas, after 60 days, 90 days and 120 days the higher percentage of viability was recorded at -196oC for both Vero cells and Avian Influenza virus.&amp;nbsp; Different pH levels were set for both samples separately with same time interval and found slightly acidic pH (pH5.5) optimum for cryopreservation of Vero cell line and Neutral pH (pH7) for Avian Influenza virus. DMSO (20%) and Glycerol (40%) showed optimum percentage viability when cryopreserved for 120 days without any ill effect.&amp;nbsp;&amp;nbsp;

P-501 Live birth rates (LBR) are not influenced by a change in embryo quality imposed by embryo cryopreservation and thawing in single euploid frozen embryo transfers.

Abstract Study question Is a change of embryo quality through vitrification and thawing associated with a reduced LBR when a single euploid frozen embryo transfer is performed? Summary answer Live birth rates are not influenced by a change in embryo quality through embryo cryopreservation and thawing in single euploid frozen embryo transfer. What is known already Embryo aneuploidy is the most significant single factor and accounts for about 50% of implantation failure and miscarriage in human reproduction. Preimplantation genetic test for aneuploidy (PGT-A) presents a mean to identify euploid embryos, thereby improving pregnancy rates and reducing early pregnancy losses. However, after PGT-A on trophectoderm cells at blastocyst stage, embryos have to be cryopreserved and warmed later for the embryo transfer procedure. Embryo quality is known to impact implantation and ongoing pregnancy rates, therefore, the aim of this analysis was to evaluate the impact of a change in embryo quality between pre-vitrification and post-thaw on the outcome. Study design, size, duration This retrospective observational study included a total of 611 pregnant patients after frozen-thawed euploid single embryo transfer (FET) at a tertiary IVF centre between April 2017 and December 2020. All embryos transferred were cryopreserved after trophectoderm biopsy for PGT-A, performed either on day 5 or day 6. Each patient was only included once into the analysis. Endometrial preparation for FET was performed either in a natural cycle (NC) or as a hormonal replacement cycle (HRT). Participants/materials, setting, methods Patients with infertility, undergoing ovarian stimulation and FET in either a HRT or a NC as endometrial preparation approach, and who achieved a pregnancy, were included. Embryos were morphologically graded pre-vitrification and post-thaw based on inner cell mass and trophectoderm morphology on the day of cryopreservation. Four groups were initially established (top, good, fair, poor), hence for the analysis, fair and poor qualities were combined. Main results and the role of chance A total of 591 patient were analysed after exclusion of 20 patients due to the occurrence of late miscarriage and ectopic pregnancy. Patients’ characteristics are as follows (mean±SD (min-max)): age 33.87±5.55(20-47) years; AMH 3.20±3.28ng/mL(0.01-35.72); BMI(body mass index) 26.61±4.86kg/m2(14.84-40.31) and AFC 14.00±7.33(1-35). The changes in the embryo quality pre-vitrification and post-thaw were categorized in “no change”, “improved” or “degraded”. All groups were compared to top-quality-no change group. A binary logistic analysis evaluated the following groups regarding their combined impact on LB: top quality-degraded (odds ratio (OR)0.702; 95% confidence interval (CI)0.302-1.635, p = 0.413); good quality-no change (OR 1.022; CI:0.457-2.287; p = 0.958); good quality-degraded (OR 0.787; CI:0.330-1.880; P = 0.590); good quality-improved (OR 0.301; 0.056-1.628; P = 0.163); fair+poor quality-no change(OR 0.521; 0.203-1.340; P = 0.176); fair+poor quality-degraded (OR 0.283; 0.056-1.438; P = 0.128) and fair+poor quality-improved (OR 1.917; 0.431-8.534; P = 0.393). No impact on the probability of LB was seen in any combined group of change in embryo quality with its pre-grades. Furthermore, no association was found for age, BMI, AMH, pre-vitrification and post-thaw quality. Embryos biopsied on day 6 instead day 5 had a decreased chance of a LB (OR 0.516; 0.305-0.875, p = 0.014). Further on, NC was associated with significantly higher chance of LB compared to HRT cycle (OR 2.629; 1.606-4.305, p = &amp;lt;0.001). Limitations, reasons for caution Limitation is the retrospective design of the study and the grading of the embryos, which might be subjective and bearing interobserver variability. Wider implications of the findings Whereas a change in the embryo quality does not impact the chance of LB after single euploid FET, a NC approach increases the odds for LB. This fact should be considered for a personalized treatment approach and in the choice of the endometrial preparation protocol. Trial registration number not applicable

Optimization of Key Technologies for Induction of Embryogenic Callus and Maturation of Somatic Embryos in Korean Pine (Pinus koraiensis)

Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low induction and proliferation rates, hindering the production of improved plant materials. We investigated the effects of the explant sterilization method, 4 °C cryopreservation, basal medium, ethylene removal, liquid medium supplementation, and a combination of PGRs on embryogenic callus (EC) induction of Korean pine, using immature embryos of Korean pine as explants. The effects of sucrose and maltose on EC proliferation and maturation were investigated. The differences in the maturation ability of EC somatic embryos before and after cryopreservation were evaluated using the induced embryonic cell lines. The results showed that zygotic embryos (ZEs) performed better than megagametophytes (MGs) as explants. The induction rate of EC was significantly increased after 28 days of cryopreservation at 4 °C. The induction rate of EC in the #5 family increased from 10.00% to 62.8%. The EC induction rate of the five families cultured with the DCR basal medium was higher than that with the mLV basal medium. Among them, the induction rate of the #5 family cultured with the mLV basal medium was 23.3%, while that with the DCR basal medium was 60.9%, an increase of 2.6 times. There was no significant difference in the maturation ability of EC somatic embryos before and after cryopreservation. In conclusion, this study provides a method to improve the EC induction rate and maturation ability of Korean pine.

The effectiveness of adding apple cider vinegar and garlic to chicken meat kebabs as an antimicrobial and its role in improving its sensory and physiochemical properties

Making apple cider vinegar with garlic puree added to it and studying the effect of its antimicrobial, organoleptic and physiochemical properties on chicken thigh meat prepared for making kebabs under refrigeration. The chicken thigh meat was immersed in apple cider vinegar and garlic at concentrations of (50, 70, 100)%, while the control sample was immersed in distilled water. The meat samples were placed in polyethylene bags and kept in refrigeration at 5°C for 11 days. Some organoleptic tests such as color, flavour, taste, general acceptability and microbial tests were performed and included The total number of bacteria, coliform bacteria, proteolytic bacteria, and the physiochemical tests of moisture, pH, and total volatile nitrogen، during the storage period, the effectiveness of vinegar with garlic puree against some bacterial species was tested by diffusion method, Pseudomonas florescence , Salmonella typhimurium, Escherichia coli , Staphylococcus aureus. The results showed a decrease in microbial numbers after the seventh day of cryopreservation, compared to the number after the first day, for all bacterial species examined and for all concentrations used. It was observed that their numbers increased after the eleventh day of preservation, indicating spoilage and microbial spoilage of the kebabs. An improvement in the sensory characteristics and for all concentrations was observed with the vinegar used compared with the control model. and a decrease in the humidity and pH levels was noted, as well as an increase in the percentage of total volatile nitrogen on the eleventh day, indicating the start of proteolysis and the deterioration of the kebab product during that period. The results showed a variable effect on the inhibition ability according to the concentration used and the type of bacteria tested, which increased with the increase in the concentration used, and the highest inhibition diameter was 29 mm towards Staph bacteria. aureus at 50% concentration. The lowest inhibition diameter was 10 mm against E.coli and Pseudo. florescence at concentration 30 %.

Cryopreservation of Pig Semen Using a Quercetin-Supplemented Freezing Extender.

Reactive oxygen species (ROS) produced during freeze–thaw procedures cause oxidative damage to the sperm, reducing fertility. We aimed to improve the post-thaw quality of pig sperm by quercetin (QRN) supplementation to reduce the cryodamage associated with the freeze–thaw procedure. Four equal aliquots of pooled boar semen were diluted with a freezing extender supplemented with different concentrations of QRN (0, 25, 50, and 100 µM) and then were subjected to cryopreservation in liquid nitrogen. Semen analysis was performed following 7 days of cryopreservation. Results demonstrated that the semen samples supplemented with 50 µM QRN significantly improved the post-thaw sperm quality than those subjected to other supplementations (p < 0.05). Semen samples supplemented with 50 µM QRN showed significantly improved plasma membrane functional integrity (47.5 ± 1.4 vs. 43.1 ± 4.1, 45.3 ± 1.7, and 44.1 ± 1.4) and acrosome integrity (73.6 ± 3.4 vs. 66.3 ± 2.4, 66.7 ± 3.6, and 68.3 ± 32.9) as compared to the control, 25 µM, and 100 µM QRN groups, respectively. The mitochondrial activity of the 50 µM QRN group was greater than control and 25 µM QRN groups (43.0 ± 1.0 vs. 39.1 ± 0.9 and 41.9 ± 1.0) but showed no difference with the 100 µM QRN group. Moreover, the 50 µM QRN group showed a higher sperm number displaced to 1 cm and 3 cm points in the artificial mucus than other groups. Therefore, supplementing the freezing extender with QRN can serve as an effective tool to reduce the magnitude of oxidative damage associated with sperm freezing.

Effect of Adding α-tocopherol on Fertility Parameters in Bovine Semen Cryopreservation

Background: Cryopreservation of bovine semen affects survival of spermatozoa. It is necessary to improve semen quality to increase fertility. The aim was to determine the effect of α-tocopherol added to the bovine semen diluent at different time of freezing, upon sperm quality post thawing. Methods: Semen was collected from Charolais breed bulls. The semen was divided into two portions and one portion was added with α-tocopherol and the other was used as control. These portions were further divided into three parts and frozen for 8, 11 and 15 minutes, respectively. After 60 days of cryopreservation, the progressive motility, non-progressive motility, absence of motility, vitality and DNA fragmentation were evaluated. Result: The lower percentages of DNA fragmentation were treated with antioxidants (15.52, 14.83 and 14.71%). The highest vitality and progressive motility were found in the sample with α-tocopherol and frozen for 11 min with 72.91 and 58.61%, respectively. The addition of α-tocopherol to the semen diluent decreased damages to spermatozoa and enhanced the semen quality.

Влияние переносов размороженных бластоцист, экспандированных на пятые или шестые сутки на эффективность программ ВРТ

Повышение эффективности программ вспомогательных репродуктивных технологий (ВРТ) невозможно без совершенствования и улучшения условий культивирования эмбрионов с последующим переносом или криоконсервацией экспандированных бластоцист достигших нужной стадии к пятым или шестым суткам развития.Цель исследования - оценка влияния дня культивирования бластоцист человека, достигших необходимой стадии экспандирования на эффективность программ ВРТ, частоту наступления клинической беременности (ЧКБ), частоту ранних потерь и частоту живорождения в криопротоколах при проведении программ ВРТ. Проанализированы данные программ переноса размороженных эмбрионов (ПРЭ) экспандированных на 5-е, группа А (N=2275) или 6-е сутки, группа Б (N=170). Частота наступления беременности (ЧНБ) в исследуемых группах составила 50,8 и 46,5%, соответственно. Не наблюдалось статистически достоверных различий в показателях частота наступления клинической беременности (ЧКБ) 37,4 и 37,0% и частота ранних потерь беременности 26,0 и 21,5% у пациенток обеих групп, соответственно. Основной показатель эффективности работы клиники ЭКО – частота живорождения (Take-HomeBabyRate) в криопротоколах с переносами экспандированных бластоцист на пятые (группа А) или шестые сутки (группа Б) составил 36,5 и 35,2%, соответственно (разница не достоверна).В криопротоколах день экспансии бластоцист (5-е или 6-е сутки развития) статистически не влияет на ЧКБ, частоту ранних потерь беременности и частоту живорождения. В программах ПРЭ в первую очередь следует отдавать приоритет качеству бластоцист (отличное и хорошее) не зависимо от дня их криоконсервации.

Protein profile as a quality indicator of cryopreserved semen from Tambaqui Colossoma macropomum (Cuvier, 1818).

The aim of this study was to evaluate the association between proteins in the seminal plasma of tambaqui Colossoma macropomum (Cuvier, 1818) with seminal quality indicators after thawing. The semen was cryopreserved with a dilution based on BTS with 8% DMSO. A 200 µL sample of semen from each animal was diluted in 800 µL BTS, centrifuged at 800 rpm, and the supernatant was cryopreserved to further analyze of the protein profile of seminal plasma through one-dimensional electrophoresis (SDS-PAGE). After 15 days of cryopreservation, a cryopreserved semen straw was thawed to analyze both qualitative and quantitative parameters. When considering all collections, the SDS-PAGE identified 15 protein bands in the seminal plasma of tambaqui. When the interaction (presence or absence) between proteins observed in the seminal plasma and the post thawed spermatic parameters was evaluated, we observed a great influence of the presence of proteins on spermatic quality. A greater (P<0.05) fertilization rate was observed with the presence of proteins 12, 34, 44, 85, and 90 kDa. Proteins in seminal plasma of tambaqui influenced the spermatic quality after thawing, and thus, they can be utilized as an indicator of sperm quality, especially the proteins with a molecular weight ≤ 50 kDa.

Blasts from the past: is morphology useful in PGT-A tested and untested frozen embryo transfers?

Research questionDay of cryopreservation, inner cell mass (ICM) grade, trophectoderm grade and blastocyst expansion grade have been associated with differences in live birth rate in frozen embryo transfer (FET) cycles. This study sought to examine the likelihood of live birth and whether the morphological grade of the blastocyst is more or equally useful in FET cycles among preimplantation genetic testing for aneuploidies (PGT-A) tested and untested blastocysts. DesignThis was a retrospective cohort study of 6271 vitrified-warmed, autologous, single-embryo transfer cycles among patients undergoing IVF from July 2013 to December 2017 at a single, university-affiliated infertility practice. The primary outcome was live birth, calculated by generalized estimating equations. ResultsAmong PGT-A tested embryos, inferior ICM grade was associated with a lower chance of live birth (ICM grade B versus A: adjusted risk ratio [aRR] 0.91, 95% confidence interval [CI] 0.84–0.99). Among untested blastocysts there was a lower live birth rate in blastocysts cryopreserved on day 6 versus day 5 (aRR 0.87, 95% CI 0.78–0.96), and those with inferior pre-vitrification trophectoderm grade (trophectoderm grade B versus A: aRR 0.86, 95% CI 0.79–0.94). Blastocysts with a higher pre-vitrification expansion grade (pre-vitrification expansion grade 5 versus 4: aRR 1.1, 95% CI 1.01–1.2) were associated, but ICM grade was not associated (ICM grade B versus A: aRR 0.93, 95% CI 0.86–1.02), with chance of live birth. ConclusionsAmong PGT-A untested blastocysts, assessing embryo quality by day of cryopreservation, trophectoderm grade and expansion grade may help to identify embryos with the highest likelihood of live birth. Identifying euploid embryos by PGT-A appears to homogenize the cohort, making blastocyst morphological grade and day of cryopreservation less important.

Dynamics of the Level of Myeloperoxidase and Serum Calprotectin in Local Cold Injury

Relevance. Cold injury is defined as a complex of pathophysiological and pathomorphological changes arising as a result of local or general cooling of the human body. Endothelial dysfunction provokes a powerful cascade of cellular interactions with expressed excretory activity, which ultimately leads to a pronounced remodeling of microcirculation and a protracted process of inflammation in the focus of alterations. Aim of the study. To establish the dynamics of the level of myeloperoxidase and calprotectin in the serum of patients with local cold injury. Materials and methods. The study included 80 patients with III–IV degree lesions in the late reactive period and the period of granulation and epithelialization. The average age of patients was 38 ± 8 years. The myeloperoxidase level was measured on the 5th and 30th days from the moment of cryopreservation using multiplex analysis of blood serum. Results. The level of myeloperoxidase and serum calprotectin increases. In late reactive period (day 5), MPO level in patients with frostbite is 7.25 times higher in comparison with control values, in the period of granulation and epithelialization (day 30), it remains elevated, but only 3.63 times higher than in the control group. In the late reactive period, the level of calprotectin in the blood serum of patients with local cold injury was 4.6 times higher in comparison with control values, and on the 30th day of cryopreservation, the value of calprotectin was 4.5 times higher than in the control group. Changes in the level of myeloperoxidase and serum calprotectin reflect the flow of destructive and reparative mechanisms in tissues during local cold trauma and can be used in predicting an unfavorable prolonged course of the wound process.

Gelatin carrageenan sericin hydrogel composites improves cell viability of cryopreserved SaOS-2 cells

Cryopreservation and the low revival rate of cryopreserved cells remains a major challenge in cell based bone regeneration therapies. In our current study we aimed to develop a sericin based hydrogel composite incorporating various drugs and growth factors to enhance cell attachment, cryopreservation to increase the cellular viability upon revival. Sericin, gelatin and carrageenan blended hydrogel composites were prepared and explored for their physicochemical properties. The hydrogels prepared were porous and showed higher biocompatibility. Further, silver nanoparticles, alendronate and insulin like growth factor (IGF-1) were incorporated into the hybrid hydrogels individually and checked for sustained drug release profile. IGF-1 incorporated hydrogels composites showed better osteogenic cell attachment, proliferation and cell revival upon cryopreservation. The clonogenic potential of seeded cells upon 30 days of cryopreservation was also evaluated which was 55% in IGF-1 incorporated scaffold cells. A flow cytometry based staining protocol using Annexin V was developed which showed a live cell population up to 80% even after 30 days of crypreservation. These results validate the potential of our formulated hydrogels as cell based systems aimed for increasing cell survival upon cryopreservation and thus has a great potential for bone repair and regeneration.

Feasibility of a cryopreservation of cultured human corneal endothelial cells.

Transparency of the cornea is essential for vision and is maintained by the corneal endothelium. Consequently, corneal endothelial decompensation arising from irreversible damage to the corneal endothelium causes severe vision impairment. Until recently, transplantation of donor corneas was the only therapeutic choice for treatment of endothelial decompensation. In 2013, we initiated clinical research into cell-based therapy involving injection of a suspension of cultured human corneal endothelial cells (HCECs), in combination with Rho kinase inhibitor, into the anterior chamber. The aim of the present study was to establish a protocol for cryopreservation of HCECs to allow large-scale commercial manufacturing of these cells. This study focused on the effects of various cryopreservation reagents on HCEC viability. Screening of several commercially available cryopreservation reagents identified Bambanker hRM as an effective agent that maintained a cell viability of 89.4% after 14 days of cryopreservation, equivalent to the cell viability of 89.2% for non-cryopreserved control cells. The use of Bambanker hRM and HCECs at a similar grade to that used clinically for cell based therapy (passage 3–5 and a cell density higher than 2000 cells/mm2) gave a similar cell density for cryopreserved HCECs to that of non-preserved control HCECs after 28 days of cultivation (2099 cells/mm2 and 2111 cells/mm2, respectively). HCECs preserved using Bambanker hRM grew in a similar fashion to non-preserved control HCECs and formed a monolayer sheet-like structure. Cryopreservation of HCECs has multiple advantages including the ability to accumulate stocks of master cells, to transport HCEC stocks, and to manufacture HCECs on demand for use in cell-based treatment of endothelial decompensation.

Cat ovarian follicle ultrastructure after cryopreservation with ethylene glycol and dimethyl sulfoxide

Ovarian tissue cryopreservation is a promising technique for fertility maintenance. The aim of this study was to compare the morphology of domestic cat ovarian follicles after tissue cryopreservation with ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). Ovaries from healthy adult cats undergoing elective ovariohysterectomy were used. Eight fragments were obtained from each pair of ovaries: two were used as fresh controls; three were submitted to fresh perfusion toxicity test and perfused with M199, 10% fetal calf serum and 0.4% sucrose containing Me2SO 1.5 M, EG 1.5 M or Me2SO 0.75 M + EG 0.75 M; and the remaining three fragments were perfused as described and submitted to slow freezing. After 45 days of cryopreservation, the samples were thawed, fixed and processed for light and transmission electron microscopy (TEM). The percentages of morphologically normal follicles identified by light microscopy were higher in the control group (94.45%) in comparison to the frozen groups (80.56% with EG, 78.7% with Me2SO and 75.87% with EG + Me2SO). The fresh perfused tissue showed no statistical difference compared to control or frozen samples. The TEM analysis showed less damage in the ultrastructure of follicles from the Me2SO group in comparison with the EG and Me2SO + EG groups. According to the morphological analysis, 1.5 M Me2SO is the best cryoprotectant for cryopreservation of domestic cat ovarian tissue regarding the morphology of preantral follicles after thawing. Further studies regarding the viability of these follicles should be performed.

Successful application of a single warming protocol for embryos cryopreserved by either slow freezing or vitrification techniques

ABSTRACTThe aim of this study was to evaluate the feasibility and efficiency of using a sucrose gradient-based warming protocol as a universal warming approach on human cleavage stage embryos. Between January 2013 and November 2014, a total of 118 warming cycles were performed on 705 embryos which had previously been cryopreserved/thawed by slow freezing protocols or cryopreserved by slow freezing and warmed by vitrification thaw solution. Clinical outcomes have been retrospectively analyzed depending on cryopreservation and warming techniques used, embryo viability, day of cryopreservation, clinical pregnancy, implantation, and live birth rate. Results indicate that, the use of the vitrification warming protocol for warming after slow freezing results in comparable post-warming survival (71.6% and 71.1%; p = 0.890). Higher clinical pregnancy, implantation, and live birth rates were obtained in the cryopreserved embryos by slow freezing and warmed by vitrification group in comparison to the cryopreserved/thawed by slow freezing protocols group but the results did not show statistically significant differences between groups (p > 0.05). These results indicate that such an approach can eliminate the need to search for a brand-dependent product, as well as case-dependent hands-on planning. Further research that evaluates the effectiveness of this approach on a larger case series is underway.Abbreviations: CPA: concentrated cryoprotective agent; COH: controlled ovarian stimulation; FET: frozen embryo transfer; HSG: hysterosalpingogram; mHTF: modified human tubal medium; SSM: single step media; SSS: synthetic serum substitute; TV-USG: transvaginal ultrasound.

Are pregnancy rates affected by day of blastocyst cryopreservation in single euploid frozen embryo transfer cycles?

There is inconclusive evidence whether day of cryopreservation of euploid embryos affects pregnancy rates. We sought to compare pregnancy outcomes between euploid blastocyst embryos cryopreserved on either day 5 or 6. Retrospective cohort study. 126 cycles performed at a large university-based center during a period of January 2013 through December 2016 were evaluated. Euploid embryos cryopreserved on either day 5 (n=77) or day 6 (n=51) and utilized in a single frozen embryo transfer (FET) cycle were included for analysis. Preimplantation genetic screening was performed by either array Comparative Genomic Hybridization or Next-Generation Sequencing. Only high quality blastocyst embryos were biopsied and frozen. The primary outcome was ongoing pregnancy rate (OPR). Secondary outcomes included implantation rate (IR), clinical pregnancy rate (CPR), clinical loss rate (CLR) and multiple gestation rate (MGR). Student’s t-test and Mann-Whitney test were used to assess continuous variables depending on how the data was distributed. Chi-square was used to assess categorical variables. Multivariate logistical regression was performed to control for potential confounding variables. A p-value <0.05 was considered statistically significant. No significant differences were noted between the groups for age (35.5±4.3 vs. 35.5±4.0), BMI (25.8±5.8 vs. 25.2±4.6), AMH level (3.1±2.3 vs. 3.6±4.4), previous live birth (37.7% vs. 49.0%), diagnosis of recurrent pregnancy loss (RPL) (23% vs. 14%) and number of prior FET cycles (0.3±0.7 vs. 0.6±0.9). There were no differences in pregnancy outcomes between the two groups (Table). Using bivariate analysis there was no difference in OPR between the two groups (OR: 0.84, 95% CI 0.40 - 1.73, p=0.63). A multivariate regression analysis was performed to control for the potential confounding variables of age, BMI, previous live birth, diagnosis of RPL, or prior FET resulting in an adjusted odds ratio for OPR of 0.78 (95% CI 0.37 - 1.65, p=0.51). Day of cryopreservation does not affect pregnancy outcomes in single euploid blastocyst transfers. Rate of multiples remains low in single embryo transfer cycles.Tabled 1Pregnancy OutcomesDay 5Day 6p-valueIR, n (%)55/77 (71.4)36/51 (70.6)nsCPR, n (%)53/77 (68.8)35/51 (68.6)nsOPR, n (%)45/77 (58.4)32/51 (62.7)nsCLR, n (%)8/53 (15.1)3/35 (8.6)nsMGR, n (%)1/53 (1.9)2/35 (5.7)nsns=not significant Open table in a new tab

Cryopreservation of rat hepatocytes with disaccharides for cell therapy

Cryopreservation of hepatocytes is a crucial step in the implementation of cell therapy for treating certain liver diseases. In the present study we investigated the effect of the some disaccharides on the cryopreservation of rat hepatocytes. Liver cells were frozen in media in the presence or absence of low concentrations of Me2SO (5% Me2SO) supplemented with varying concentrations of disaccharides (sucrose, glucose and trehalose). After 7 days of cryopreservation, the hepatocytes were thawed and viability was measure by exclusion of trypan blue and by the MTT technique, as well as by determining albumin production. Among the investigated disaccharides and concentrations, 0.2 M trehalose showed the best overall outcome. Compared to the use of Me2SO alone, significant improvement in post-thaw cell viability was observed. The new solution may reduce Me2SO side effects on patients and improve the viability and quality of cryopreserved hepatocytes.

EXTRACTIUON AND LYOPHILIZATION OF THE LOW DENSITY OF LIPOPROTEIN INSTEAD OF EGG YOLK ON SOME SEMEN PARAMETERS OF AWASSI RAMS

This study was conducted to explain the possibility of lyophilization of low– density lipoprotein (LDL) that is extracted from the egg yolk to be used instead of the egg yolk in an extender semen that belong to the Awassi sheep and their effect on the characteristics of the semen after five days of cryopreservation, as well as to get low-density Lipoprotein, steriled and lyophilized (powder) lipoprotein that can be stored for a long time to used when needed. The study was conducted in the from July 2015 to February 2016. The first stage started from July until August 2015 the (LDL) was extracted from fresh egg. The second stage in August 2015. the liquid (LDL) has been lyophilized and turn into powder that is packed in vials, steriled vials and then was stored in 0°C. The third stage was conducted from January to February 2016 in the college of Agriculture/ University of Baghdad. the semen was collected from four of the Awassi rams of the age (2– 4) years and the samples was collected by using artificial vagina. After that the sample was divided on the experimental treatments evenly 1ml/ transaction by using extender Tris. The dilution ratio was 1: 10. The effect of the different concentrations had been studied of the LDL on the characteristic of semen and three concentration of the LDL were used (3.2, 4.8 and 6.2%) for the transactions T1, T2,T3 respectively and the control treatment (20% EY).The effect of the lyophilized LDL was studied on some characteristic of sperm when cryopreserved under the temperature 5°c for five days. The Results of the experiment to varying differences between transactions in sperm characteristics as treatment outperformed T3 (P &lt;0.05) in the individual movement in the 2nd and 3rd days and did not notice a significant difference on the other days, while a significant difference in the percentage of live sperm among the treatments and control were observed viability of keeping LDL of sperm higher was than it is in the control treatment of the plasma membrane as treatment outperformed T2 and T3 (P &lt;0.05) on the treatment of control on the 3rd, 4th and 5th of conservation was also excel themselves transactions in susceptibility saved for acrosome on different days of conservation compared with the control treatment as it overtook treatment T2 in the first two days and did not notice a significant difference on the 3rd and 4th among the treatments and control excelled treatments T2 and T3 (P &lt;0.05) in 5th day of conservation to the control, we conclude from the foregoing that it is possible freeze-drying LDL and save the freeze and use it in time of need can also be used in lyophilized LDL thinners semen because of its positive impact in keeping sperm has been observed that the best concentration of sperm in maintaining the properties is 6.4% LDL compared with egg yolk and other transactions.

Preparation of Internal Quality Control Material for Lymphocyte Subset Analysis.

Lymphocyte subset analysis is widely used in clinical laboratories, and more than two levels of daily QC materials are required for reliable results. Commercially available, expensive QC materials have short shelf lives and may not be suitable in resource-poor settings. We compared different methods for preparing homemade QC material, including fixation with 1%, 2%, or 4% paraformaldehyde (PFA); freezing with 10% dimethylsulfoxide (DMSO), 0.1% bovine serum albumin-phosphate buffered saline, or after ethanolic dehydration; and using cryopreservation temperatures of -20℃, -80℃, or -196℃. We found an optimal experimental condition, which is 'fixation with 4% PFA, freezing with 10% DMSO, and storage at 80℃'. To evaluate long-term stability of QC materials prepared in this optimal condition, two levels of QC materials (QM1 and QM2) were thawed after 30, 33, 35, 37, 60, 62, 64, and 67 days of cryopreservation. Lymphocyte subset was analyzed with BD Multitest IMK kit (BD Biosciences, USA). QM1 and QM2 were stable after 1-2 months of cryopreservation (CV <3% for CD3, CD4, and CD8 and 5-7% for CD16/56 and CD19). We propose this method as an alternative cost-effective protocol for preparing homemade internal QC materials for lymphocyte subset analysis in resource-poor settings.