Brian C. Su

Human papillomavirus gene sequences in washed human sperm deoxyribonucleic acid

The present study demonstrated the presence of HPV gene sequences in Percoll-washed sperm cells using polymerase chain reaction primers targeting smaller gene regions. Up to 64% of the sperm specimens were shown to contain gene sequences indicative of the presence of HPV. Human papillomavirus type 16 was detected about twice as often as HPV type 18. The results suggest the possible role of sperm as a vector for HPV.

White blood cells in semen affect hyperactivation but not sperm membrane integrity in the head and tail regions

The presence of high numbers of peroxidase-positive PML in ejaculated semen significantly reduced sperm HA, an important step leading to sperm capacitation. Sperm membranes at both the head and tail regions, as assessed by the hypo-osmotic viability parameter and the hypo-osmotic sperm swelling test, respectively, were not affected by peroxidase-containing leukocytes. Sperm motility was not affected, but sperm curvilinear and straight line velocity parameters were reduced in the presence of high concentrations of leukocytes in the ejaculate. The results suggested that the effect of leukocytes on sperm was through a reduction in sperm hyperactive motility but not through alterations in the sperm head and tail membranes.

Assessment of sperm for cryopreservation using the hypoosmotic viability test

In summary, the hypoosmotic viability parameter was significantly correlated with the outcome of the thawed sperm motility. The prefreeze supravital staining for sperm viability and the hypoosmotic sperm swelling test were not predictive of the thawed sperm total motility. The hypoosmotic viability parameter was not correlated to the postwarmed sperm motility after refrigeration. The results indicated that the integrity of the sperm membranes at the head were more important than the tail membrane.

Sperm hyperactivation as quality control for sperm penetration assay

The sperm penetration assay (SPA) is subject to considerable variation, and controls are needed to verify the accuracy of the results. It is proposed that sperm hyperactivation (HA) can serve as a quality control check for the SPA. The objective was to determine if there was an association between the SPA outcome and sperm HA measured at various times during the SPA procedure. The data showed a significant correlation between percent sperm HA and percent zona-free oocyte penetration by sperm preincubated for three hours prior to sperm-oocyte interaction (short preincubation). Some sperm hyperactivity was observed in liquefied raw semen samples, but this was insignificantly related to SPA results. Low correlation was observed between SPA results and sperm HA determined immediately after centrifuge washing of sperm. The results suggest that it is possible to utilize sperm HA measured immediately after the sperm-oocyte interaction period as a quality control check of SPA results.

A double method sperm wash for artificial insemination

Recently, there have been concerns regarding the presence of reactive oxygen species (ROS) produced during sperm processing for insemination. However, the sperm wash methods that yielded low ROS levels also had low sperm recovery after processing. The objective of this study was to compare sperm recovery after swim-up from pellet, overlay, and 2-layer Percoll wash methods with the recovery after the double method wash. The latter method consisted of a combination of 2 sperm wash methods, namely, the overlay and the Percoll method. Motile sperm were first collected through the overlay method. The leftover semen was then processed through the 2-layer Percoll method to scavenge motile sperm and the resultant pellet combined with the pellet from the overlay method. In this manner, the level of ROS was kept to a minimal, sperm recovery was improved, and a mixture of sperm with different surface properties was produced as a result of using different processing methods. The results indicated an improvement in sperm recovery and in total sperm motility in noncryopreserved sperm after using the double method wash when compared with the other wash methods. The study suggests that the double method wash is a feasible method for processing sperm for insemination.

Diadenosine Tetraphosphate (Ap4A) and Triphosphate (Ap3A) Signaling of Human Sperm Motility

The ubiquitous dinucleotide polyphosphate, diadenosine tetraphosphate (Ap4A), has been shown to be a signal molecule for DNA replication in mammalian cells. In this study, Ap4A and a related compound, diadenosine triphosphate (Ap3A), were tested for possible signaling functions in human spermatozoa. A computerized automated semen analyzer was used to detect changes in spermatozoa motility parameters. Cryopreserved-thawed donor spermatozoa were washed and incubated in 0.1 mM Ap4A, 0.1 mM Ap3A, or control medium. The data indicated that both Ap4A and Ap3A decreased the percentage of motile spermatozoa after 4 or more hours of incubation in vitro. The two dinucleotide polyphosphates caused an increase in the amplitude of lateral spermatozoa head displacement parameter only at the start of incubation. The other spermatozoa kinematic parameters were unaffected. No opposing ying-yang dual actions of Ap4A to Ap3A were seen. From the results, Ap4A and Ap3A were observed to be potential inhibitory signals of spermatozoa motility after prolonged exposure.

Uptake of exogenous human papilloma virus L1 DNA by oocytes and detection by the polymerase chain reaction

ObjectiveThe purpose of this study was to determine if oocytes were capable of taking up exogenous DNA such as human papillomaviral (HPV) DNA and evaluate the zona pellucida as a barrier to the entry of foreign DNA into the oocyte.MethodsThe experiment consisted of four groups of hamster oocytes exposed to HPV DNA fragments: Group A, zona-free oocytes (n =5); Group B, oocytes with an intact zona pellucida (n =5); Group C, oocytes fixed in 4% buffered formalin solution for 20 min (n =5); and Group D, zona-free oocytes (n =4). Group C oocytes served as an internal control to ensure adequate washing of the oocytes after incubation.ResultsThe zona pellucida was not a barrier to foreign DNA molecules. The PCR did not detect L1-HPV and β-globin gene sequences in the untreated hamster oocyte. Uptake of the smaller DNA fragments such as that amplified from the β-globin region was independent of active oocyte cell processes.ConclusionOocytes cultured in vitro can passively take up exogenous DNA fragments. The results suggest a possible role of oocytes as vectors for foreign DNA.

Clinical pregnancy rate after the double method wash and intrauterine insemination.

The protocol for intrauterine insemination (IUI) involves sperm processing using different methods that have produced varying results. These sperm wash methods do not take into consideration the problems of the exact timing of ovulation and the requirements of sperm cells at different stages of capacitation. The objectives of this study were 1) to use the double method wash previously reported to produce a mixture of different populations of sperm cells and determine the pregnancy outcome after IUI and 2) to compare the sperm kinematic parameters after the double method wash with those after the centrifuge (or whole-population) wash method. Patients were divided into either the double method group (n = 119) or the centrifuge method group (n = 76). The Hamilton-Thorn HTM-C automated sperm motility analyzer (Hamilton-Thorn Research, Danvers, MA) was used to analyze sperm motility parameters. Pregnancy outcomes were evaluated after controlled ovarian stimulation and IUI. An almost 2-fold increase was seen in the pregnancy rate with the double method wash compared with the centrifuge method wash. Sperm motility and velocity were also enhanced in the double method wash groups. The results support the usefulness of the double method wash for the preparation of sperm for IUI.