Virginia N. Bolton

Development of spare human preimplantation embryos in vitro: An analysis of the correlations among gross morphology, cleavage rates, and development to the blastocyst

Following in vitro fertilization, the criteria commonly used to select human embryos for transfer are the cleavage rate and gross morphology, the contention being that those embryos which divide more rapidly and have regular, spherical blastomeres are more likely to lead to a pregnancy. In order to assess the validity of this assumption, the development in vitro of spare embryos was investigated. Eggs and embryos were cultured in Earles balanced salt solution containing 10% heat-inactivated patients serum, and insemination was performed at 40 hr post human chorionic gonadotropin (hCG). At 82–90 hr post hCG, up to four embryos were transferred. Any spare embryos were cultured in the same medium for up to 6 days and scored daily for cell number and morphology using a “quality” scale of 4-1 according to degree of fragmentation and shape of the blastomeres. Of 317 fertilized eggs, 55 (17%) developed to the fully expanded blastocyst stage. The remaining embryos ceased development at the one-cell (6; 2%), two-cell (49; 15%), fourcell (110; 35%), eight-cell (61; 19%), and cavitating morula (36; 11%) stages. The relationship between developmental arrest and gross morphology is discussed.

Preimplantation genetic diagnosis as a novel source of embryos for stem cell research.

The generation of human embryonic stem (hES) cells has captured the public and professional imagination, largely due their potential as a means of overcoming many debilitating and degenerative diseases by cell replacement therapy. Despite this potential, few well-characterized hES cell lines have been derived. Indeed, in the UK, despite several centres having been active in this area for more than 2 years, there are as yet no published reports of human embryonic stem cells having been generated. Part of the reason for this lack of progress may relate to the quality of embryos available for research. Embryos surplus to therapeutic requirements following routine assisted reproduction treatment are often of poor quality and a large proportion may be aneuploid. This study reports a new approach to hES cell derivation. Embryos surplus to therapeutic requirements following preimplantation genetic diagnosis were used. Although unsuitable for embryo transfer due to the high risk of genetic disease, these embryos are from fertile couples and thus may be of better quality than fresh embryos surplus to assisted reproduction treatment cycles. Embryos donated after cryopreservation were also used, and putative hES lines were derived from both sources of embryos. The cell lines described here are thought to be the first reported hES cell lines to have been derived in the UK.

Pregnancies after in vitro fertilization and transfer of human blastocysts

In a study of 29 cycles of IVF, ET was performed on day 5 after oocyte recovery when embryos had developed to the morula/blastocyst stage. Three preclinical pregnancies and three live births resulted (2 singleton and 1 twin), giving a viable PR per ET of 10%. It is concluded that while day 5 ET may well be important in terms of embryo biopsy for the preimplantation diagnosis of genetic disease, day 2 ET remains preferable for therapeutic IVF. Although these data would not support the introduction of day 5 ET into routine therapeutic IVF, delayed ET should be considered as an alternative approach to preimplantation diagnosis. Indeed, because the latter will generally involve the treatment of normal, fertile couples, it might be predicted that embryo survival rates, and thus the rate of pregnancy after day 5 ET, would be better than those presented here.

Imprinted Expression of SNRPN in Human Preimplantation Embryos

Summary Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two clinically distinct neurogenetic disorders arising from a loss of expression of imprinted genes within the human chromosome region 15q11-q13. Recent evidence suggests that the SNRPN gene, which is defective in PWS, plays a central role in the imprinting-center regulation of the PWS/AS region. To increase our understanding of the regulation of expression of this imprinted gene, we have developed single-cell–sensitive procedures for the analysis of expression of the SNRPN gene during early human development. Transcripts of SNRPN were detected in human oocytes and at all stages of preimplantation development analyzed. Using embryos heterozygous for a polymorphism within the SNRPN gene, we showed that monoallelic expression from the paternal allele occurs by the 4-cell stage. Thus, the imprinting epigenetic information inherited in the gametes is recognized already in the preimplantation embryo. The demonstration of monoallelic expression in embryos means that efficient preimplantation diagnosis of PWS may be made by analysis for the presence or absence of SNRPN mRNA.

Pregnancies following in vitro fertilization and ultrasound-directed surgical embryo transfer by perurethral and transvaginal techniques

Ultrasound-directed surgical ET is useful in patients with a history of difficult cervical (nonsurgical) transfer, and was performed without complications in the small group reported here. The technique is straightforward, and requires no greater expertise than that necessary for ultrasound-guided oocyte collection. However, further studies are necessary to assess its role in the routine transfer of embryos following IVF.

Chapter 5 Development of The Human Preimplantation Embryo In Vitro

Publisher Summary This chapter discusses the development of human preimplantation embryo in vitro. The successful fertilization in vitro of human eggs and the in vitro culture of human preimplantation embryos has become a daily challenge for hundreds of laboratories throughout the world. The factors that influence the success of in vitro fertilization (IVF) include the uterine microenvironment, embryo replacement technique, culture conditions, and egg or embryo quality. Most therapeutic IVF units use superovulatory regimes with clomiphene citrate, human menopausal gonadotrophin, pure follicle-stimulating hormone, or a combination of these hormones, in order to achieve multiple folliculogenesis. The media used for culture of human eggs and embryos in vitro have been adapted from those derived originally for culture of animal embryos, primarily the mouse preimplantation embryo. As a consequence of therapeutic regimes that induce the formation of multiple follicles, many patients yield more embryos than are required for replacement. In most therapeutic IVF units, embryos are replaced into the recipients uterus between 40 and 48 hours after insemination.

Laparoscopic zygote intrafallopian transfer using augmented local anesthesia

In this study, 29 laparoscopic ZIFTs were performed in 21 patients using local anesthesia augmented with intravenous analgesia. The technique was well tolerated; significant discomfort arose only when the fallopian tubes were manipulated and was minimized by transferring zygotes to one tube only. Seven pregnancies resulted, of which three have delivered and one is ongoing.

Retrograde ejaculation: a systematic approach to non‐invasive recovery of spermatozoa from post‐ejaculatory urine for artificial insemination

Summary. Retrograde ejaculation is an uncommon but treatable form of male infertility. Successful recovery of live spermatozoa from the post‐ejaculatory urine for artificial insemination is dependent on careful regulation of pH and osmolarity of the urine into which ejaculation takes place, and separation of the motile spermatozoa from the debris and cells which are found in these samples. Three pregnancies established by artificial insemination of spermatozoa recovered by non‐invasive means from the bladders of men suffering from retrograde ejaculation are described. The techniques for preparing the urine for spermatozoal survival, and for removal of cells and debris by sedimentation or buoyant density centrifugation are discussed.

Assisted hatching is more effective when embryo quality was optimal in previous failed IVF/ICSI cycles

Summary Assisted hatching (AH) was developed as a possible solution to repeated implantation failure. The aim of this analysis was to examine the relationship between the morphology of embryos in a previous cycle on outcome in a subsequent cycle with AH. A total of 175 AH cycles performed after previous failed ART without hatching were divided into group A with optimal and group B with suboptimal embryos transferred previously. The groups were similar in terms of demographic and cycle characteristics. In group A, there was a significant improvement (p < 0.001) in implantation (28.8 vs 5.1%), clinical pregnancy (41.9 vs 12.1%) and live birth rate (38.5 vs 8.6%) compared with group B. The data suggest that the prognosis for treatment is better if AH is performed after failure despite optimal embryos compared with failure associated with suboptimal embryos and embryo quality is the most significant factor affecting outcome.

The effects of lignocaine on human sperm motility

ObjectiveThe purpose of this study was to assess the motility of human sperm incubated with various concentrations of lignocaine.MethodsEleven semen samples with a sperm density ⩾20 ×106ml and progressive motility ⩾40% were prepared using a swim-up technique. Aliquots from each sample were incubated for 4 hr under capacitating conditions with lignocaine concentrations of 100, 10, 1, and 0.1 µg/ml and without additional lignocaine as a control. Digital computerized motion analysis was performed on all samples at 1, 2, and 4 hr after the addition of lignocaine.ResultsAfter 2 hr of incubation a significant (P <0.05) increase in the percentage of sperm with a curvilinear velocity >100 µm/sec was observed in those samples incubated with 100 µg/ml lignocaine. This stimulatory effect was no longer apparent after a further 2 hr of incubation. No other significant changes were identified in any of the motility parameters examined.ConclusionsNo adverse effects on human sperm motility were identified during incubation with low concentrations of lignocaine. A transient stimulatory effect was observed at a lignocaine concentration of 100 µg/ml.