Cryoprotectant-free vitrification of human spermatozoa in new artificial seminal fluid

Vitrification is a new method that has been recently introduced in Assisted Reproduction Technique programs. The aim of this study was to design a new medium similar to normal human seminal fluid (SF), formulation artificial seminal fluid (ASF), and to compare the cryoprotective potency of this medium with SF and human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with the swim-up technique and sperm suspensions were divided into four aliquots: (i) fresh sample (control); (ii) vitrification in HTF medium supplemented with 5 mg/mL human serum albumin and 0.25 mol sucrose (Vit HTF); (iii) vitrification with patients' SF (Vit SF); and (iv) vitrification in ASF (Vit ASF). After warming, sperm parameters of motility, viability, and morphology were analyzed using WHO criteria. Also, sperm pellets were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy and transmission electron microscopy observations. The results showed that progressive motility (46.09 ± 10.33 vs. 36.80 ± 13.75), grade A motility (36.59 ± 11.40 vs. 16.41 ± 11.24), and normal morphology (18.74 ± 8.35 vs. 11.85 ± 5.84) and viability (68.22 ± 10.83 vs. 60.86 ± 11.72) of spermatozoa were significantly higher in Vit ASF than in Vit HTF. All parameters were better in Vit ASF than in Vit SF, but only viability was significantly different (p = 0.006). After cryopreservation, deep invagination in cytoplasm and mechanically weak point sites and folded tail were commonly observed. But, this phenomenon was more significant in Vit HTF and Vit SF than in ASF (p < 0.05). In transmission electron microscopy evaluation, acrosome damage, plasma membrane loss, chromatin vacuolation, and disruption of mitochondria arrangement and structures were observed in all vitrified groups. Adherence of several tail sections together was also seen in all cryo groups. But this was seen more in Vit HTF and Vit SF than in ASF (p < 0.05). In conclusion, vitrification of human spermatozoa with ASF can effectively preserve the quality of sperm motility in comparison with Vit HTF. © 2016 American Society of Andrology and European Academy of Andrology

Validation of air freezing index (AFI), for determination of frost penetration depth in typical arid and semi-arid zones of Iran

Depth of frost penetration is one of the main indices in agriculture, civil and transportation engineering. Soil temperature is a function of several factors including: topography, solar radiation, air temperature, moisture content and other physical properties of soil such as thermal capacity, coefficient of heat conductivity, and specific heat. The main objective of the present paper is to determine the frost penetration depth in soils based on the air temperature. In this study the daily and hourly temperatures of air and soil at different depths of three climatology stations located at Shahr-e-Kord, Yazd and Urmia cities of Iran were collected and analyzed for a period of 11 years from 1992 to 2003. In the first stage, Air Freezing Indices (AFI) of the three named stations was calculated using three methods known as: American, Norwegian and Finn and then the results were compared with the observed values accordingly. Investigations showed that correlation between the results is significant at one percent level, but the three methods gave different figures. Based on other references, it has been shown that the American method is more suitable for regions located at the middle latitudes. Thus, the correlations between Frost Penetration Depth (FPD) and AFI based on the US method were calculated and found to be 0.88 and 0.82 for Shahr-e-Kord and Urmia stations respectively. However, the correlation for Yazd station was much lower (0.65), and significant (P<0.05). As a general, it was concluded that application of this method is more relevant to semi-arid rather than arid zones, but in the absence of a better method, the same index could be used for determination of FDP in arid zones as well

Erratum: Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017

Interpretation: By quantifying levels and trends in exposures to risk factors and the resulting disease burden, this assessment offers insight into where past policy and programme efforts might have been successful and highlights current priorities for public health action. Decreases in behavioural, environmental, and occupational risks have largely offset the effects of population growth and ageing, in relation to trends in absolute burden. Conversely, the combination of increasing metabolic risks and population ageing will probably continue to drive the increasing trends in non-communicable diseases at the global level, which presents both a public health challenge and opportunity. We see considerable spatiotemporal heterogeneity in levels of risk exposure and risk-attributable burden. Although levels of development underlie some of this heterogeneity, O/E ratios show risks for which countries are overperforming or underperforming relative to their level of development. As such, these ratios provide a benchmarking tool to help to focus local decision making. Our findings reinforce the importance of both risk exposure monitoring and epidemiological research to assess causal connections between risks and health outcomes, and they highlight the usefulness of the GBD study in synthesising data to draw comprehensive and robust conclusions that help to inform good policy and strategic health planning

Impact of different embryo loading techniques on pregnancy rates in in vitro fertlizaton/embryo transfer cycles

Background: Embryo transfer (ET) technique is one of the important factors of in vitro fertlization success. Among the different steps in ET technique, less attention has been given to embryo loading (EL). The aim was to compare the impact of two different techniques of EL on pregnancy rate in IVF/ET cycles. Materials and Methods: In this retrospective study, 144 and 170 patients were placed in groups A and B, respectively. In Group A, the embryos were drawn directly into the ET catheter from culture microdrop under the oil. In Group B, the embryos were transferred from culture microdrop into G2 medium in center-well dish. Then, the embryos were drawn into the catheter and finally transferred into the uterus. Both groups were adjusted for other parameters based on the EL technique. The main outcome measure was pregnancy rate. Results: There were insignificant differences for etiology of infertility, source of sperm, type of stimulation protocol, percent of IVF or intracytoplasmic sperm injection type of ET catheter, cycles with good quality embryos and transferred embryos between two groups. The rate of both chemical and clinical pregnancy was higher in Group B compared to A, but the difference was insignificant (P = 0.09 and P = 0.1, respectively). Conclusion: It seems that there is no difference in the outcome by loading the embryo from microdrop or center-well dish

Morphology and viability of human spermatozoa vitrified with a new, cryoprotectant-free, artificial seminal fluid

Cryopreservation is a process finalized to store tissues and cells at a very low temperature. The most common freezing protocols used for gamete preservation in Assisted Reproductive Technologies are slow freezing and vitrification (1). Vitrification combines ultrarapid cooling with high concentrations of cryoprotectants; it avoids, better than slow freezing, the formation of ice crystals. It has been demonstrated, however, that cryoprotectant addition may significantly reduce cell viability (2). This study was aimed to design a new, cryoprotectant-free, medium similar to normal human seminal fluid (SF) formulation (artificial seminal fluid; ASF), and to compare the cryoprotective potential of this medium with SF and Human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with swim-up technique and sperm suspensions were divided in four groups: fresh (controls); vitrified in HTF (Vit HTF); vitrified in patients’ SF (Vit SF); and vitrified in ASF (Vit ASF). To identify the effects of the different media we assessed sperm parameters of motility, viability and morphology after warming. Spermatozoa ultrastructure was also evaluated by scanning and transmission electron microscopy (SEM and TEM). The results showed that sperm motility, viability and normal morphology were significantly higher in Vit ASF than in Vit HTF. The same parameters were better in Vit ASF than in Vit SF, but only viability differed significantly. Deep cytoplasmic invaginations and folded tails were commonly observed by SEM in all vitrified sperms, but this alterations were more evident in Vit HTF and Vit SF than in Vit ASF. By TEM, acrosome damage, plasma membrane loss, chromatin vacuolation, disruption of mitochondria and adherence of several tail sections together were observed in all vitrified groups; the latter phenomenon, however, was more evident in Vit HTF and Vit SF than in Vit ASF. In conclusion, vitrification of human spermatozoa with ASF seems more effective in preserving sperm quality than Vit SF and, particularly, Vit HTF

Efficacy of the in vitro splitting of human preimplantation embryos from ART programs

Background/aim: The aim of this study was to evaluate the efficiency of in vitro embryo splitting (IES) procedures. We also assessed the quality of the blastocysts developed from embryos obtained from different sources. Materials and methods: Good quality embryos at 6–8-cell stages were categorized according to their fertilization sources: 1) frozenwarmed donated embryos, 2) chromosomally abnormal embryos, 3) parthenogenetic embryos, and 4) embryos derived from fertilization of in vitro matured oocytes (rescue IVM). After IES, splitting and developmental efficiency was assessed. Furthermore, the quality of the developed blastocysts was evaluated by Hoechst and propidium iodide (PI) staining. Results: The data showed a high rate of both splitting and developmental efficiency in the frozen-warmed embryos after IES (140% and 71.7%, respectively), followed by chromosomally abnormal embryos (96.8% and 52.5%, respectively). Results of the Hoechst and PI staining showed that the mean ± SD cell numbers of the control group were higher (113.11 ± 16.01) than that of twins A (donor blastomeres embryos, 58 ± 12.2) and B (recipient blastomeres embryos, 50.4 ± 8.5), respectively. Conclusion: Chromosomally normal embryos enrolled in IES are more potent to develop into viable blastocysts. For research purposes, 1PN and 3PN embryos are the best options for splitting procedures, regardless of the poor quality of developed blastocysts

Embryo cryopreservation following In-vitro maturation for fertility preservation in a woman with Mullerian adenosarcoma: A case report

In-vitro maturation (IVM) of the immature oocytes recovered from the surgically removed ovarian tissue has been considered as a process for fertility preservation in patients with cancer. Fertility preservation for a woman with Mullerian adenocarcinoma. A 37-year-old woman with Mullerian adenocarcinoma was a candidate for ovarian resection. The immature oocytes were retrieved after ovarian resection from a 37-year-old woman with Mullerian adenocarcinoma. The oocytes underwent IVM and were fertilized using intracytoplasmic sperm injection (ICSI). Two healthy embryos were cryopreserved for future use. The immature oocytes from the ovarian tissue can be matured with IVM for generation of embryos after ICSI. The embryos can be vitrified using routine methods for fertility preservation in young women with cancer

Human cumulus cell sensitivity to vitrification, an ultrastructural study

Abstract Cumulus cells (CCs) play an important role in the regulation of female gamete development, meiotic maturation, oocyte-sperm interaction, capacitation and acrosome reaction. However, their role in maintaining oocyte competence after vitrification is unclear as controversial data on their protecting action against oocyte cryoinjuries are available. Here we described the effects of vitrification on the ultrastructure of human CCs collected from women undergoing assisted reproductive technologies (ARTs). In total, 50 patches of CCs, sampled from high-quality human cumulus-oocyte complexes, were randomly allocated into two groups after patient informed consent: 1, fresh CCs (controls, n = 25); 2, vitrified CCs (n = 25). Samples were then prepared and observed by transmission electron microscopy. In fresh CCs, in which small cell clusters were visible, cell membranes were joined by focal gap junctions. Microvilli were rare and short. Nuclei, mitochondria, smooth endoplasmic reticulum (SER), Golgi apparatus and lipid droplets appeared well preserved; vacuoles were scarce. After vitrification, we observed two populations of CCs: light CCs, with a smooth appearance and few short microvilli; and dark CCs, with numerous and long microvilli. In both, most of the organelles appeared similar to those of fresh CCs. Lipid droplets were denser and more numerous, with respect to fresh CCs. They were mainly located in the peri-nuclear and sub-plasmalemmal regions. Numerous packed electron-negative vacuoles were visible. The vitrification procedure did not cause alterations in the fine structure of major organelles, except for an increased amount of lipid droplets and vacuoles. This specific sensitivity of human CCs to vitrification should be considered during ARTs

The Exact Synchronization Timing Between the Cleavage Embryo Stage and Duration of Progesterone Therapy-improved Pregnancy Rates in Frozen Embryo Transfer Cycles: A Cross-sectional Study

Background: Synchronization between the embryonic stage and the uterine endometrial lining is important in the outcomes of the vitrified-warmed embryo transfer (ET) cycles. Objective: The aim was to investigate the effect of the exact synchronization between the cleavage stage of embryos and the duration of progesterone administration on the improvement of clinical outcomes in frozen embryo transfer (FET) cycles. Materials and Methods: 312 FET cycles were categorized into two groups: (A) day- 3 ET after three days of progesterone administration (n = 177) and (B) day-2 or -4 ET after three days of progesterone administration (n = 135). Group B was further divided into two subgroups: B1: day-2 ET cycles, that the stage of embryos were less than the administrated progesterone and B2: day-4 ET cycles, that the stage of embryos were more than the administrated progesterone. The clinical outcome measures were compared between the groups. Results: The pregnancy outcomes between groups A and B showed a significant differences in the chemical (40.1% vs 27.4%; p = 0.010) and clinical pregnancies (32.8% vs 22.2%; p = 0.040), respectively. The rate of miscarriage tended to be higher and live birth rate tended to be lower in group B than in group A. Also, significantly higher rates were noted in chemical pregnancy, clinical pregnancy, and live birth in group A when compared with subgroup B2. Conclusion: Higher rates of pregnancy and live birth were achieved in day-3 ET after three days of progesterone administration in FET cycles. Key words: Endometrium, Embryo transfer, Pregnancy, Live birth, Progesterone