The first frozen embryo donation pregnancy for hypergonadotrophic hypogonadism in Singapore--hormonal profile and obstetric outcome

This is the first report in South East Asia of a singleton frozen embryo donation pregnancy for hypergonadotrophic hypogonadism. The hormonal profile was compared with that of a control group of normal uncomplicated singleton pregnancies in Singapore. The plasma beta hCG levels were lower compared to those of our normal uncomplicated singleton pregnancies at 2 to 3 weeks after the embryo transfer but became comparable at 4 to 5 weeks after embryo transfer. The successful vaginal delivery and the obstetric complications developed in this case are discussed.     

A Three Dimensional Anchorage Independent In Vitro System for the Prolonged Growth of Embryoid Bodies to Study Cancer Cell Behaviour and Anticancer Agents

We describe a three dimensional (3D) anchorage independent in vitro protocol for the prolonged growth of human embryoid bodies (EBs) up to 90 days. We grew hESCs (46XX) in methylcellulose (MC) in motion culture in the presence of EB medium (EB), EB medium with Matrigel (EB + MAT), bulk culture medium (BCM), and BCM medium with Matrigel (BCM + MAT). All four experimental groups produced embryoid bodies (EBs) which with prolonged growth to 90 days acquired blood vessels and tissues from all three germ layers. Based on histology, microarray gene expression profiles and the definition for experimental teratomas, we could classify the EBs into early EBs, mature EBs and teratomas. The EB + MAT group produced the highest number of teratomas and their microarray data suggested the presence of inductive microenvironment niches and activation of pathways for self-organization, morphogenesis and growth. When we microinjected hepatocarcinoma-Green Fluorescent Protein cells (HepG2-GFP) (46XY) into the teratomas, after 10 days the HepG2-GFP cells had grown inside the teratoma as confirmed by confocal microscopy and SRY gene analysis. This 3D-MC-(EB + MAT) in vitro system requires few cells to produce many teratomas, can be used to test pluripotency of potential human embryonic and induced pluripotent stem cell lines (hESC, hiPSC), and is an experimental humanized platform to study cancer cell behavior.     

An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells

Human embryonic stem cells (hESCs) promise to revolutionize reparative medicine through their potential in developing cell replacement therapies for diseases like diabetes and parkinsonism. Most of the existing hESC lines available for research, including all National Institutes of Health-registered lines, have been derived and maintained on mouse embryonic fibroblast feeders in the presence of xenoproteins. For future clinical application, many more hESC lines derived and grown in current good manufacturing practice, good tissue culture practice, and xeno-free conditions need to be developed. Concurrently, effective cryopreservation methods that prevent or limit the accidental contact of hESCs with nonsterile liquid nitrogen during periods of long-term storage have to be formulated. We describe a safe, xeno-free cryopreservation protocol for hESCs involving vitrification in closed sealed straws using human serum albumin as opposed to fetal calf serum as the main protein source in the cryoprotectant and long-term storage in the vapor phase of liquid nitrogen. After thaw, hESCs exhibited high thaw-survival rates and low differentiation rates, remained pluripotent, and maintained normal diploid karyotypes throughout extended passage. The cryopreservation technique we describe here should complement xeno-free culture conditions for hESCs already in refinement and will prove very useful for the setting up of hESC banks throughout the world.     

Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in-vitro fertilization and understanding implantation

It has been shown recently that delayed transfers improve implantationrates in assisted reproductive technology programmes. In a prospectivestudy, the pregnancy rates and safety of outcome were evaluatedin a group of patients after the transfer of day 5 blastocystswith enzymatic treatment of the zona pellucida. Nineteen womenwith a mean age of 32.6 ± 5.2 years and mean 2.1 ±2.2 repeated attempts had blastocyst transfers with a mean numberof 2.5 ± 0.7 embryos replaced per patient. The clinicalpregnancy rates per cycle/transfer and implantation rate were53% and 33%, respectively. The multiple pregnancy rate was 40%(two pregnancies were triplets). The pregnancy and implantationrates were very much higher than observed for most assistedreproduction technology centres. The ‘in-vitro implantation’rates of zona-free blastocysts on a variety of feeder monolayerswas 92%, offering some thoughts as to the role of the zona andinteraction of the inner cell mass and trophoectoderm with theendometrium in implantation. Based on the in-vitro studies andthe high multiple pregnancy rates, it appears that zona-manipulatedblastocysts implant relatively well and there would be a needto reduce the number of transferred embryos to one or two, thusreducing multiple pregnancies and having spare blastocysts availablefor cryopreservation. The results also suggest that using theembryo culture protocol and method of transfer in the presentstudy offers encouraging improvements to assisted reproductiontechnology, and enzymatic treatment of the zona may allow betteranchorage and dialogue of the embryo with the endometrium, helpingus to improve and understand implantation.     

Ultrastructural observations of enzymatically treated human blastocysts: zona-free blastocyst transfer and rescue of blastocysts with hatching difficulties

Enzymatic treatment of the zona pellucida to either soften or remove totally the zona before blastocyst transfer has resulted in high implantation rates. The zona is usually completely dissolved after 1.5 min exposure with 10 IU pronase at 37 degrees C. Since there may be concerns that pronase treatment for periods of 1.5 min or longer may cause adverse effects on the trophectoderm (TE) and inner cell mass (ICM), the changes to human blastocysts exposed to different time intervals of pronase were investigated. Of 18 blastocysts exposed to pronase for 1.5 min, the zona was completely dissolved and no changes were observed by light microscopy (LM) or transmission electron microscopy (TEM), compared with 11 naturally hatched untreated blastocysts (controls). In another five blastocysts exposed to pronase for 2 min, no LM changes were observed but subtle TEM changes such as fewer bundles of tonofibrils attached to desmosomes were observed. When three other blastocysts were exposed to pronase for 5 min, the blastocoele collapsed, and the TE cells started to show blebbing under LM. Under TEM, the cytoplasm of TE cells was extensively vacuolated and many TE cells showed cytoplasmic blebbing towards the blastocoele. However, the epithelium was uninterrupted with intact tight junctions and desmosomes. Of a separate group of 44 blastocysts cultured in vitro, 54.5% had hatching difficulties when monitored from day 5 to day 8 and 80% of these could be rescued by removal of the zona with pronase for 1.5 min prior to extensive degeneration taking place. The results confirm that the optimal time for softening or complete removal of the zona before transfer was around 1.5 min and that enzymatic treatment was a safe, non-invasive procedure to remove the zona of blastocysts. The human embryonic TE is a very hardy, robust epithelium that withstands pronase treatment.     

The evaluation of various culture media in combination with dimethylsulfoxide for ultrarapid freezing of murine embryos.

Bicarbonate-buffered HTF medium, Medicult, and T6 are as effective as PB1 medium when used in combination with DMSO in ultrarapid freezing of two-cell mouse embryos. However, the use of phosphate-buffered T6 results in reduced in vitro development and inner cell mass size as compared with bicarbonate- and Hepes-buffered T6 when used with 3.5 M of DMSO. Hence, the use of this media for ultrarapid freezing should be avoided when this concentration of DMSO is used.     

Micro-insemination: genetic aspects

Micro-insemination involves sperm deposition directly into oocytes. This can be by transfer of sperm (Micro-Insemination Sperm Transfer, or MIST) or by micro-injection into the ooplasm (Micro-Insemination Micro-Injection into Cytoplasm, or MIMIC). Micro-insemination is indicated in spermatozoa with no or very poor motility, very low density, multiple defects, or inability to penetrate oocyte vestments. There is a 10% incidence of chromosomal abnormalities in spermatozoa from fertile and normal men. However, there is no increase in sperm chromosomal abnormalities in men with normal peripheral karyotypes and highly abnormal sperm parameters. Preliminary results of karyotypes of human oocytes that failed to become fertilized after MIST and mouse morulae and blastocysts produced after MIST reveal that there was no significant increase in aneuploidy or polyploidy. There is evidence that MIMIC may result in increased abnormal sperm karyotypes. Polyspermy is low in the mouse and human after transfer of multiple spermatozoa into the perivitelline space, thus suggesting an oolemmal block. However, blastomere membranes do not fuse with spermatozoa, as observed in a study of MIST into human embryos. Zona drilling with acid is not advised because of disturbances to chromosomal kinetics. The conclusion of this review is that MIST does not result in an increased risk of chromosomal abnormalities, while caution must be exercised with MIMIC.     

Comparative silver staining patterns of water buffalo, goat, and pig spermatozoa

Spermatozoa from river and swamp water buffalo (Bubalus bubalis), goat, and pig were stained using a silver nitrate procedure and examined under bright field optics. The silver nitrate differentiated many detailed morphologic features of the head, midpiece, and tail of spermatozoa between the species studied. Acrosomal integrity due to sperm injury or aging and various sperm abnormalities were also clearly identified by silver nitrate. Silver staining patterns revealed species-specific and strain-specific differences, particularly of the sperm head. The biochemical basis of silver staining has been attributed to the presence of sulphydryl and disulphide-rich proteins. The technique is relatively inexpensive, rapid, and repeatable and may be useful for biological research and evaluation of semen for artificial insemination.