No abstract

Ooplasmic transplantation aimed at restoring normal growth in developmentally compromised oocytes and embryos was evaluated in seven couples (eight cycles) with multiple implantation failures. Two approaches were investigated to transfer ooplasm from donor eggs at metaphase II (MII) stage into patient MII eggs: (i) electrofusion of a ooplasmic donor fragment into each patient egg (three cycles), and (ii) direct injection of a small amount of ooplasm from a donor egg into each patient egg (five cycles). Some donor eggs were used multiple times. Donor eggs were divided into two groups, one being used for ooplasmic extraction and the other one for egg donation. Cleaved embryos resulting from the latter were cryopreserved, where numbers and satisfactory development permitted. A second control group consisted of embryos derived from patient eggs after intracytoplasmic sperm injection without ooplasmic transfer. This was performed when sufficient number of eggs were available (n = 5). Donor eggs (n = 40) were evaluated cytogenetically after micromanipulation in order to confirm the presence of chromosomes. One egg was anuclear and the recipient embryos were not transferred. Normal fertilization was significantly higher after injection of ooplasm (63%) in comparison with fusion (23%). Pronuclear anomalies appeared enhanced after fusion with ooplasts. Embryo morphology was not improved in the three cycles with electrofusion and patients did not become pregnant. An improvement in embryo morphology was noted in two patients after injection of ooplasm and both became pregnant, but one miscarried. A third pregnancy was established in the repeat patient, without obvious embryo improvement. One baby was born and the third pregnancy is ongoing with a normal karyotype. Two other patients with male factor infertility had poor embryos after ooplasmic injection, but the donor embryo controls were also poor. The patients did not become pregnant and had no donor embryos frozen. Ooplasmic transfer at the MII stage may be promising in patients with compromised embryos; however, evaluation of ooplasmic anomalies and optimization of techniques will require further investigation prior to widescale application.

Incidences of chemical air contamination (CAC) are common in assisted reproductive technology, but not reported in peer review format. Justified fear of car and industrial emissions clearly exists among reproductive specialists, but standards for air contents and gaseous emission limits have not been reported. Here, we describe air sampling methods and assay systems which can be applied to any laboratory or laboratory item. It was found that unfiltered outside air may be cleaner than high efficiency particulate air filtration (HEPA) filtered laboratory air or air obtained from incubators, due to accumulation of volatile organic compounds derived from adjacent spaces or specific laboratory products such as compressed CO2, sterile Petri dishes and other materials or devices known to release gaseous emissions. Specific groups of products such as anaesthetic gases, refrigerants, cleaning agents, hydrocarbons and aromatic compounds such as benzene and toluene are described. The latter were shown to accumulate specifically in incubators. Isopropyl alcohol was the most dominant product found, though it was not used by the laboratory staff. Concentrations of this agent were low in incubator air, indicating that it was probably absorbed by the water in the pan or by culture medium. Measures to counter CAC are proposed, including the use of activated carbon filters and oxidizing material placed in the central air handling systems, in separate free-standing units or even inside the incubators.

A procedure is described that allows cryopreservation and efficient post-thaw recovery of either a single or a small group of human spermatozoa. This is achieved by injecting them into cell-free human, mouse or hamster zonae pellucidae before the addition of cryoprotectant. The method involves a combination of physical micromanipulation procedures and glycerol-mediated cryoprotection. Zonae were tracked by positioning them in straws between two small air bubbles prior to freezing. Spermatozoa from poor specimens were cryopreserved and their fertilizing ability after thawing was compared with that of fresh spermatozoa from fertile men. Human eggs used for fertilization testing were either 1 day old or in-vitro matured. Only 2% of the frozen zonae were lost and >75% of spermatozoa cryopreserved in this manner were recovered and prepared for intracytoplasmic sperm injection. The feasibility of cryopreserving a single spermatozoon was assessed. Fifteen motile spermatozoa were frozen in 15 zonae, of which 14 were recovered after thawing. Ten were injected into spare eggs, of which eight became fertilized. Spermatozoa recovered mechanically from human zonae fertilized the same proportion of oocytes as fresh fertile control spermatozoa. The recovery and fertilization rates with spermatozoa frozen in animal zonae were 87 and 78% respectively. The fertilization rate was marginally higher (P < 0.05) than that for spermatozoa frozen in human zonae, perhaps because the latter may have acrosome reacted more frequently. The zona pellucida appears to be an ideally suited sterile vehicle for storage of single spermatozoa.

Testing shows that most laboratories conducting human gamete and embryo culture have air quality and sources of contamination that exceed the levels measured in homes, businesses and schools. The sources of these contaminants have been shown to be either from activities outside the laboratory, or emitted from materials used in the facility, such as compressed gas, cleaning and sterilizing agents, plastic and stored materials. Both the laboratory structure and the air handling systems may affect the air composition. The significance of these findings is being validated by the accumulation of field case studies and now by assay procedures. Products given off by road sealant were shown to have accumulated in one of the examined laboratories, adjacent to a large re-surfaced parking area. Aldehydes such as acrolein, hexanal, decanal, pentanal and others were detected at elevated concentrations that were statistically significant. Since it is not appropriate to add potentially suspect chemicals to human embryos, we used a mouse-model to study the effect of acrolein. The growth of mouse embryos was significantly affected after acrolein was added at different concentrations to the culture environment. The physiological effect was noted at concentrations in the low ppm range. The testing end-point of embryo death must still be considered to be a crude basis for evaluating toxicological effects, since it involves addition of compounds to culture media and unprotected growth until the blastocyst stage. The findings may, however, support observations of decreased pregnancy rate following exposure of human embryos to aldehydes or other adverse conditions. With proper engineering and material selection, it is possible to reduce such contamination. The usefulness of this approach for controlling aldehydes has been demonstrated by decreasing levels in the laboratory to below those of the outside air.