Live births achieved via IVF are increased by improvements in air quality and laboratory environment.

Infertility is a common disease, which causes many couples to seek treatment with assisted reproduction techniques. Many factors contribute to successful assisted reproduction technique outcomes. One important factor is laboratory environment and air quality. Our facility had the unique opportunity to compare consecutively used, but separate assisted reproduction technique laboratories, as a result of a required move. Environmental conditions were improved by strategic engineering designs. All other aspects of the IVF laboratory, including equipment, physicians, embryologists, nursing staff and protocols, were kept constant between facilities. Air quality testing showed improved air quality at the new IVF site. Embryo implantation (32.4% versus 24.3%; P < 0.01) and live birth (39.3% versus 31.8%, P < 0.05) were significantly increased in the new facility compared with the old facility. More patients met clinical criteria and underwent mandatory single embryo transfer on day 5 leading to both a reduction in multiple gestation pregnancies and increased numbers of vitrified embryos per patient with supernumerary embryos available. Improvements in IVF laboratory conditions and air quality had profound positive effects on laboratory measures and patient outcomes. This study further strengthens the importance of the laboratory environment and air quality in the success of an IVF programme.

Laser-assisted zona pellucida thinning does not facilitate hatching and may disrupt the in vitro hatching process: a morphokinetic study in the mouse.

STUDY QUESTION: Does laser-assisted zona thinning of cleavage stage mouse embryos facilitate hatching in vitro? SUMMARY ANSWER: No, unlike laser zona opening, zona thinning does not facilitate embryo hatching. WHAT IS KNOWN ALREADY: Artificial opening of the zona pellucida facilitates hatching of mouse and human embryos. Laser-assisted zona thinning has also been used for the purpose of assisted hatching of human embryos but it has not been properly investigated in an animal model; thinning methods have produced inconsistent clinical results. STUDY DESIGN, SIZE, DURATION: Time-lapse microscopy was used to study the hatching process in the mouse after zona opening and zona thinning; a control group of embryos was not zona-manipulated but exposed to the same laser energy. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eight-cell CB6F1/J mouse embryos were pooled and allocated to three groups (n = 56 per group): A control group of embryos that were exposed to a dose of laser energy focused outside the zona pellucida (zona intact); one experimental group of embryos in which the zona pellucida was opened by complete ablation using the same total number of pulses as the control group; a second experimental group of embryos in which the zona pellucida was thinned to establish a smooth lased area using the same number of pulses as used in the other two groups. The width of the zona opening was 25 µm and width of the thinned area was 35 µm. Development was monitored by time-lapse microscopy. Overall treatment differences for continuous variables were analyzed by analysis of variance and pairwise comparisons using the Student t-test allowing for unequal variances, while for categorical data, a standard chi-squared test was utilized for all pairwise comparisons. MAIN RESULTS AND THE ROLE OF CHANCE: The frequency of complete hatching was 33.9% in the control group, 94.4% after zona opening, and 39.3% after zona thinning (overall group comparison, P < 0.0001). Overall, 60.7% of the zona-thinned embryos did not complete the hatching process and remained trapped within the zona; when they did hatch, they did not necessarily hatch from the zona-thinned area. Hatching in about one-third of the zona-intact embryos began with breaches at multiple sites by small groups of cells. Likewise, 53.6% of zona-thinned embryos had multiple breaches, always involving an area outside the thinned zone. Zona opening decreased multiple breaching and led to blastocyst escape an average of 14 h earlier than zona-thinned embryos and 5.5 h before control embryos (P = 0.0003). LIMITATIONS, REASONS FOR CAUTION: The experiments presented here were limited to in vitro experiments performed in the mouse. Whether human embryos would behave the same way under similar circumstances is unknown. We postulate that zona thinning is not beneficial in human embryos. WIDER IMPLICATIONS OF THE FINDINGS: The experiments demonstrate that zona thinning is not equivalent to zona opening for assisted hatching. The study provides reason for systematic reviews of assisted hatching trials to take the method of assisted hatching into consideration and not combine the results of zona thinning and zona opening procedures. STUDY FUNDING/COMPETING INTERESTS: Institutional funds were used for the study. No competing interests are declared.

Derivation of human embryonic stem cells in xeno-free conditions.

Human embryonic stem cells (hESC) have the potential to treat a wide range of diseases. Currently, the use of existing hESC lines in human clinical applications is limited, as they are derived from blastocysts subjected to immunosurgery with animal derived antibodies, and are maintained on mouse embryonic feeder (MEF) cells, in the presence of either fetal calf serum (FCS) or on Matrigel or with conditioned media from MEFs. Successful derivation of hESCs in xeno-free conditions is crucial in advancing stem cell therapy applications. Two hESC lines, one from chromosomally abnormal embryos and another cell line from normal embryos from the inner cell mass of human blastocysts are derived using a culture media that had 20% serum replacement (SR) and human FGF2 on human foreskin fibroblasts as feeder cells. Derivation and characterization of such xenofree hESCs suitable for clinical studies is described in this chapter.

Cytoplasmic fragmentation in activated eggs occurs in the cytokinetic phase of the cell cycle, in lieu of normal cytokinesis, and in response to cytoskeletal disorder.

The timing of cytoplasmic fragmentation in relation to the cell cycle was studied in mature oocytes and early cleavage stages using mouse oocytes and embryos as experimental models. The central approach was to remove the nuclear apparatus, in whole or in part, from non-activated and activated oocytes and early embryos, and follow their response during subsequent culture in vitro. Oocytes arrested in metaphase of the second meiotic division did not fragment following complete removal of the meiotic apparatus, provided they were not subsequently activated. Exposure of spindle-chromosome-complex-depleted oocytes to activation conditions immediately after enucleation led to fragmentation, although not until control embryos entered first mitosis. Delaying activation until 24 h post-enucleation led to earlier fragmentation. Enucleation of normally fertilized or artificially activated oocytes after emission of the second polar body also led to fragmentation coinciding with the first mitosis in nucleated control embryos. However, if artificially activated oocytes were prevented from completing second meiosis, by exposure to cytochalasin, and then enucleated, this universal wave of fragmentation was preceded in some cytoplasts by limited fragmentation after just a few hours in culture, and coinciding with completion of meiosis II in nucleated oocytes. Fragmentation also occurred in the second mitotic cell cycle, but it was limited to blastomeres of fertilized oocytes that were enucleated in late interphase. These results indicate that fragmentation in oocytes and early embryos, though seemingly uncoordinated, is a precisely timed event that occurs only in mitotically active cells, during the cytokinetic phase of the cell cycle, in lieu of normal cytokinesis, and in response to altered cytoskeletal organization.

Fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium.

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal development, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium (ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient mice. In parallel experiments, frozen-thawed zygotes were similarly cultured and transferred. Implantation rates for transferred embryos were high (range 66-88%), regardless of whether they had been frozen as oocytes or zygotes and whether they had been transferred to the oviduct or uterus. However, fetal development was significantly higher when two-cell embryos were transferred. With blastocyst transfer, control embryos implanted and produced a greater proportion of fetuses than did oocytes frozen in CJ2, whereas transfer at the two-cell stage resulted in similar proportions of implantation sites and fetuses. Blastocyst transfer of zygotes cryopreserved in ETFM or CJ2 produced similar fetal development rates (23.6% vs 20.0%), but when frozen-thawed zygotes were transferred at the two-cell stage the fetal development rates were higher in the ETFM group (53.3%) than in the CJ2 group (32.0%). A high proportion (46.7%) of oocytes frozen in CJ2 in a nonprogrammable freezer and plunged at -20 degrees C developed into live offspring. This study shows that in the mouse (1) oocytes frozen in CJ2 can develop into viable fetuses, (2) prolonging culture in vitro has a detrimental effect on embryo transfer outcome, and (3) CJ2 offers no advantage for zygote cryopreservation.

Zona dissection by infrared laser: developmental consequences in the mouse, technical considerations, and controlled clinical trial.

Infrared laser systems are currently being marketed for application in clinical zona pellucida dissection. However, these systems have undergone only limited animal testing and minor clinical trials that lacked proper controls. Two of these systems have been evaluated in protocols that addressed potential detrimental effects on embryonic development in the mouse. Exaggerated large openings were made in the zona pellucida of 8-16 cell mouse embryos. Embryonic development and subsequent implantation and viability were assessed. A definite negative effect on these parameters was observed following the use of one of these systems. Following this animal trial, the second system was evaluated in a clinical trial for assisted hatching and embryo biopsy. Laser dissection was directly compared with the standard zona drilling using acidified Tyrode's solution. While no significant difference was evident between the two protocols, it was felt that laser dissection presented some problems in both consistency between operators and in the efficacy of subsequent manipulations such as blastomere biopsy and fragment removal. These results argue that laser zona dissection is far from a simple technique and should be carefully evaluated before any clinical application is made.