Rajasingam S. Jeyendran

Human sperm selection by glass wool filtration and two-layer, discontinuous Percoll gradient centrifugation*†

Glass wool filtration and two-layer, discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradient centrifugation resulted in an average recovery of 50% to 70% of the progressively motile and about 50% of the hypoosmotic swelling (HOS)-positive spermatozoa. Glass wool filtration tended to be more successful than Percoll centrifugation when the ejaculates were asthenozoospermic or produced a suspect/abnormal HOS test. After selection, the acrosin activity increased approximately two- to threefold, but no significant improvement in the percentage of normal sperm forms occurred. Experiments with mixtures of untreated and frozen-thawed ejaculates confirmed that glass wool filtration is more effective in removing nonmotile and HOS-negative spermatozoa than the two-layer Percoll centrifugation technique when the percentage of these types of spermatozoa in the ejaculate is high. The simplicity of these techniques and the good recovery of apparently viable spermatozoa makes these methods more desirable than other, more complicated techniques or procedures that yield a lower recovery of motile spermatozoa.

Discordance among blastomeres renders preimplantation genetic diagnosis for aneuploidy ineffective

Purpose: To investigate the contribution of discordance among blastomeres from the same embryo in the interpretation of blastomeres biopsied from day 3 embryos.Methods: 228 IVF embryos had two blastomeres removed and fluorescent in situ hybridization (FISH) was used to detect aneuploidy of chromosomes 13, 15, 16, 18, 21, 22, X and Y. Of the 228 embryos, 102 had complete FISH results for both blastomeres.Results: When the 2 blastomeres of 102 embryos with successful FISH results were compared, 26 (25.5%) were concordant for all 8 chromosomes and 76 (74.5%) were discordant for one or more chromosomes. Among the 102 embryos, 12 (12%) were disomy in both blastomeres and 37 (36%) were disomic in all 8 chromosomes in one of the two blastomeres.Conclusion: Discordance among blastomeres from the same embryo appears to present a significant problem in interpreting results of embryos biopsied on day 3 and analyzed by FISH especially when most PGD’s are done on single blastomeres.

Comparison of thrombophilic gene mutations among patients experiencing recurrent miscarriage and deep vein thrombosis.

Problemu2002 Inherited thrombophilia has been shown to be a risk factor for cardiovascular disease including deep venous thrombosis as well as reproductive disorders including recurrent pregnancy loss. We have previously reported three out of the 10 thrombophilic mutations studied, plasminogen activator inhibitor‐1 (PAI‐1) 4G/5G, factor XIII V34L, and homozygous MTHFR C667T, correlated significantly with recurrent pregnancy loss compared with controls. This study was undertaken to compare the frequencies of nine inherited thrombophilias among women with a history of recurrent pregnancy loss with individuals experiencing deep venous thrombosis and fertile controls.

Leucospermia and the fertilizing capacity of spermatozoa.

Ejaculates obtained from men attending an in vitro fertilization (IVF) program were analyzed to determine the effect of the presence of leucocytes in semen on the ability of sperm in that semen to fertilize oocytes. The mean (+/- S.E.) concentration of leucocytes in 103 ejaculates was 0.62 X 10(6)/ml (+/- 0.09) with a range of 0.1 X 10(6) or less per ml to 7.0 X 10(6)/ml. The leucocyte concentration demonstrated a significant (p less than 0.05) but inverse relationship with the fertilizing capacity of spermatozoa. Though not statistically significant, the leucocyte concentration also demonstrated an inverse relationship to sperm motility and normal morphology. These results suggest that the presence of leucocytes in an ejaculate cannot be used as the sole indicator of the fertilizing capacity of spermatozoa in that ejaculate.

Cryopreservation of human sperm in a lecithin-supplemented freezing medium

The use of egg yolk and serum albumin as additive diluents for human sperm cryopreservation is routine. But because both diluents are of animal origin, they potentially may introduce microbial agents to the sample. To reduce the risk of contamination, the cryoprotective property of phospholipids extracted from lecithin was evaluated and found to be effective when supplemented with dimethyl sulfoxide and glycerol.

ORIGINAL ARTICLE: Comparison of Thrombophilic Gene Mutations Among Patients Experiencing Recurrent Miscarriage and Deep Vein Thrombosis

Problemu2002 Inherited thrombophilia has been shown to be a risk factor for cardiovascular disease including deep venous thrombosis as well as reproductive disorders including recurrent pregnancy loss. We have previously reported three out of the 10 thrombophilic mutations studied, plasminogen activator inhibitor‐1 (PAI‐1) 4G/5G, factor XIII V34L, and homozygous MTHFR C667T, correlated significantly with recurrent pregnancy loss compared with controls. This study was undertaken to compare the frequencies of nine inherited thrombophilias among women with a history of recurrent pregnancy loss with individuals experiencing deep venous thrombosis and fertile controls.

In vitro fertilization outcome: glass wool-filtered sperm versus swim-up sperm

At present, most in vitro fertilization (IVF) programs use a swim-up procedure for sperm preparation. This method mimics the in vivo selection of motile sperm by cervical mucus. Filtration through a column of glass wool, on the other hand, may result in recovery of more viable sperm from an ejaculate with relatively poor quality as compared with the swim-up procedure. Therefore, this study was designed to compare the sperm selected by these two methods, namely the swim-up procedure and the filtration procedure with a standardized glass wool column, with regard to the ability to fertilize human oocytes in our IVF program

The hypo-osmotic swelling test for evaluation of sperm membrane integrity.

A functional membrane is requisite for the fertilizing ability of spermatozoa, as it plays an integral role in sperm capacitation, acrosome reaction, and binding of the spermatozoon to the egg surface. The hypo-osmotic swelling (HOS) test evaluates the functional integrity of the sperms plasma membrane and also serves as a useful indicator of fertility potential of sperm. The HOS test predicts membrane integrity by determining the ability of the sperm membrane to maintain equilibrium between the sperm cell and its environment. Influx of the fluid due to hypo-osmotic stress causes the sperm tail to coil and balloon or swell. A higher percentage of swollen sperm indicates the presence of sperm having a functional and intact plasma membrane. Here, we present the detailed protocol for performing the HOS test and explain the results for interpretation.

Human chorionic gonadotropin from day 2 spent embryo culture media and its relationship to embryo development

OBJECTIVEnTo detect hCG in spent embryo culture media at day 2 after intracytoplasmic sperm injection and to assess the relationship of hCG to embryo development.nnnDESIGNnExperimental study.nnnSETTINGnFertility center and clinical diagnostic laboratory.nnnSAMPLE(S)nA total of 102 spent culture media from day 2 human embryos and corresponding unexposed media for blank control.nnnINTERVENTION(S)nThe culture media samples were tested for hCG by ELISA.nnnMAIN OUTCOME MEASURE(S)nQuantity of hCG produced by embryos and correlation with the embryos developmental status.nnnRESULT(S)nhCG was found in 93 of 102 culture media tested by enzyme-linked immunosorbent assay. The correlation analysis revealed that the concentration of hCG was independent of embryo developmental status.nnnCONCLUSION(S)nThe ability to detect hCG from day 2 spent culture media may be used as a marker for embryo competence.

Glass wool-filtered spermatozoa and their oocyte penetrating capacity

The capacity of glass wool-filtered spermatozoa to penetrate zona-free hamster oocytes was studied. As compared to prefiltered sperm samples, oocyte penetration was significantly increased. A significant increase in the penetration rate for the filtered sperm population was noted even after the sperm motility in the filtrate was adjusted with medium equal to that of the prefiltered sample. However, no significant differen es in oocyte penetration were seen between the prefiltered and the filtered sperm population when the filtered sperm samples were diluted with nonviable spermatozoa. These results show that glass wool filtration yields a sperm population with a greater penetrating capacity. It was concluded that motility alone could not account for the improved penetrability and that the removal of nonviable spermatozoa may at least, in part, be responsible for this effect.