DNA extraction

Residual ThinPrep (Cytyc Corporation, Boxborough, MA) cervical samples previously tested for HPV using the Roche line blot assay [9] were used in this study. Twenty
samples containing 24 of the 27 HPV types on the reverse line blot were selected for extraction and testing. (No samples were positive for HPV 53, 55, and 57.) Therefore a total of 21 HPV types could be evaluated (there were 22 HPV capture and signal probes minus no clinical sample containing HPV 53 leaves 21 possible HPV type answers). Samples were coded so that investigators were blinded to the presence of multiple HPV types. DNA was extractedfrom 250 µL aliquots by using the QIAmp DNA Mini Kit (QIAGEN Inc., Valencia, CA). The procedure provided with the kit was followed except that the proteinase Kdigestion was done overnight at 56°C. Following DNA elution from the column, DNA was concentrated in a Centricon Centrifugal Filter Device YM-100 (Millipore
Corporation, Bedford MA) according to manufacturer's specifications. Each DNA sample was brought to 50 µLwith the addition of ultra pure water.

Terminalogy 2

Sporadic research reports continued to investigate egg freezing principally in animal models, and occasionally in the human. However, these reports tended to underline the complications and lack of consistency between cross-species comparisons. For example, while the mouse can be a useful model it must be remembered that its eggs are only just over half the volume of the human egg, and this can have a major impact on the approach to cryopreservation in these two very different species cell types. Eventually though, driven by a series of papers published by an Australian researcher called Debbie Gook from Melbourne, clinical application of egg cryopreservation began to find favor by the middle of the nineties.Recently then, the early successes have been reproduced by others in both Italy and the USA giving rise conservatively to 10 babies from the freezing of women’s own eggs. Also at least one other baby has arisen from a clinical circumstance that is not completely unfamiliar to IVF clinics. n this case eggs had been collected during a routine IVF case, but no sperm were retrievable for insemination. So the oocytes were frozen, and donor semen was selected for a later IVF attempt. Ultimately both sets of gametes were thawed and used in a bsequent IVF procedure, which achieved a health delivery. This rather specific area of plication is of great potential benefit to infertile couples undergoing IVF
therapy where perhaps problems arise unexpectedly necessitating a halt to treatment prior to insemination of the eggs with sperm. For example, in another actual situation where an IVF couple suffered an untimely death in the family at the time of their procedure, they then chose to freeze all the eggs that had been retrieved until such time as they felt able to move forward with their therapy.All of these pregnancies and reports arose from work with frozen-thawed mature oocytes , but for one notable exception, where a pregnancy arose from an immature germinal vesicle (GV) stage egg. This may not sound to be of such great importance, but it could be that this stage of egg development may prove to be a more successful approach for egg cryopreservation. Such “young” eggs are approximately one to two days away from full maturity, and as such require further growth in the laboratory in culture after thawing. They currently appear as a by-product of less than optimal ovarian stimulation for IVF where not all eggs collected are mature. However, they survive
freezing well, and possess certain features that help to maintain their integrity during the rigors of cryopreservation. For example, their membranes are more permeable to the cryoprotectant (“antifreeze”), and their chromosomes are more conveniently and safely packaged in the nucleus protecting them from disruption. Such eggs, however, still have to undergo nuclear breakdown and full maturation before they can be fertilized, and therefore their developmental competency is not so clearly established as with fully mature oocytes that are frozen. The source of
these GV eggs, and whether they have been exposed to any external hormones may play a key role in the competency of these eggs. Harvesting of these eggs and the conditions for maturation remains to be resolved fully. But provisional studies in this area
are the first to lend credence to the possibility that immature follicles and the immature eggs inside isolated from ovarian tissue, may one day be fully grown in the laboratory outside of the body.How does Cryopreservation work, and what are the

Terminology

It is worthwhile becoming comfortable with the technical terms for several of the words that will be used interchangeably in this chapter, and in case you wish to make more comprehensive searches of the literature, such explanation of terms will improve
your level of understanding. Firstly, the rather loose term egg is usually referred to as an oocyte by biologists, which is the unfertilized female gamete (sex cell). The sperm(atozoon) is the male counterpart gamete. Cryopreservation is the very
specific term for all stages involved in the cryostorage of the oocyte, and refers to the freezing, storage and the thawing processes. Cryoprotectant is the term used to refer to the fluid that is used to place the eggs in prior to freezing, and is usually a mixture of sugars and organic chemical liquids that are designed to buffer the egg cell during the stresses of freezing and thawing.To put egg freezing into context, it is interesting to consider that human sperm (spermatozoa) have been successfully frozen for decades, and that the first successful report of human embryo freezing that generated a pregnancy was in 1983. Subsequently both human sperm and embryo cryopreservation have become considered routine and consistent technologies.
Frustratingly human egg freezing has not yet reached such apparent levels of acceptance or consistency.The technology so far applied clinically has been based directly on traditional human embryo cryopreservation protocols, and has produced relatively few offspring when compared with human embryo cryopreservation. ortunately to date, no abnormalities have been reported from these pregnancies, regardless of the persistent concerns that freezing and thawing of mature oocytes may disrupt the chromosomal apparatus in these cells (meiotic spindle), and so increase the potential for chromosome abnormalities (aneuploidy) in the embryos that arise from such eggs. With respect to cryostorage of donated oocytes, for use eventually as eggs to donate to recipient women, there have been several reports that have shown some success with
this approach. In fact, it has been reported that there have been 10 babies born from frozen-thawed donor oocytes. In another unusual case, frozen donor eggs after thawing have been used, not for whole egg donation, but actually for ooplasmic transfer which
gave rise to the successful delivery of a twin following thawed ooplasmic donation. In this procedure the cytoplasm of the egg was injected into the eggs of another woman. The cytoplasm is the part of the egg outside of the nucleus that does not
contain the genetic elements (chromosomes), but contains elements of cellular unctioning. Transfer of this is thought to bolster the quality of the recipient egg making it healthier, and possibly more likely to give rise to a healthy embryo.
The first successful cryostorage of women’s own oocytes occurred with the reporting of three births over a decade ago by two centers in Australia and
Germany. However, at that time reports of egg freezing studies in mice suggested that although eggs could survive freezing and thawig, they might possess higher levels of chromosomal anomalies following this procedure when compared to fresh eggs. Of note, is the fact that these studies were not performed on human tissue, and the procedures used were totally different from that utilized with the successful human egg freezing cases.Nevertheless, the suspicion of this problem was enough to prompt a sort of voluntary worldwide moratorium on clinical oocyte cryopreservation, until studies prove the fear of chromosomal abnormalities to be unsubstantiated. Unfortunately for oocyte freezing research, human embryo cryopreservation was just starting to be undertaken much more routinely and successfully in the mid-eighties, due to the
growing ethical concerns with the fate of surplus embryos following in vitro fertilization (IVF). This presented a pressing clinical problem that drove embryo freezing research, while egg freezing languished without any clear clinical need, worsened still by the worries over its safety.