Theresa Kalugdan

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.

Evidence for ease of transmission of human papillomavirus DNA from sperm to cells of the uterus and embryo

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Transfection of the inner cell mass and lack of a unique DNA sequence affecting the uptake of exogenous DNA by sperm as shown by dideoxy sequencing analogues

Purpose: The purpose of this study was to determine whether exogenous DNA internalized into blastocysts after transference from DNA-carrier sperm are localized at the inner cell mass or trophoblast cells and to identify differences in uptake of exogenous DNA fragments by sperm due to unique DNA sequences.Methods: Mouse blastocysts at the hatching stage were exposed to migrating human sperm cells carrying exogenous DNA fragments synthesized from the E6–E7 conserved gene regions of human papillomavirus (HPV) types 16 and 18. After an interaction period of 2 hr, the transfected blastocysts were washed several times to remove extraneous sperm and the blastocysts were dissected into groups of cells derived from the inner cell mass and trophoblasts. The cells were analyzed by polymerase chain reaction (PCR) for the presence of HPV DNA fragments. In the second part of the experiment, thawed donor (N=10) sperm cells were pooled, washed, and divided into two fractions. The first (control) fraction was added with formalin and further divided and added with a35S-radiolabeled G, A, T, or C sequencing mixture. The second fraction was similarly treated but the formalin step was omitted from the treatment. After an hour of incubation at 37°C, the sperm specimens were washed several times by centrifugation and DNA extracted by the GeneReleaser method. The extracted DNA were processed on sequence gels, and the autoradiographs analyzed.Results: Mouse blastocysts transfected by carrier sperm with DNA from HPV types 16 and 18 showed localization of the HPV DNA to both the inner cell mass and trophoblast cells. Negative controls consisting of untreated human sperm and untreated mouse blastocysts did not reveal any evidence of HPV DNA. The positive sperm control generated expected DNA fragments from HPV types 16 and 18. In the second experiment, the intensities of the DNA fragments in the G, A, T, and C columns from low to high molecular weights were not different from the positive control bands. Band intensities of the four sequencing columns were similar. Formalin pretreatment of the sperm inhibited uptake of the DNA fragments from the smallest to the largest DNA molecules.Conclusions: Exogenous DNA taken into blastocysts are localized to both the inner cell mass and trophoblast cells. Only live sperm exhibited the capacity to carry various sizes of exogenous DNA, suggesting the involvement of active cell membrane mechanism in the transference process. The results showed that DNA fragments terminating in any of the four nucleotides were equally taken up by the sperm cell. Fragments of DNA produced by the sequencing reaction failed to identify a unique DNA sequence that would facilitate or inhibit the sperm from taking up exogenous DNA.

Detection of exogenous DNA in blastocysts after continuous exposure to DNA carrier sperm.

Recently, it was demonstrated that sperm cells could be used to carry foreign DNA or exogenous DNA fragments through an artificial reproductive tract and transfer the DNA to mouse blastocysts at the other end of the tract (1). Using radiolabeling methods, the DNA fragments have been reported to be localized at the postacrosomal and equatorial regions of the sperm head (2,3). There is some evidence of internalization of the DNA fragments into the compact sperm head (3). However, the majority of the foreign DNA pieces appears to be localized externally on the membrane surface and the exchange of DNA fragments between the sperm plasma membrane and the plasma membrane of hatched blastocyst cells proceeded with fluidity (1). This represents a potential problem in vivo for the inadvertent transfer of viral and bacterial DNA from the ejaculated sperm to the preimplantation blastocysL This is particularly of concern when the blastocyst is still in the reproductive tract or uterus of a patient in the early stages of pregnancy. In this study, the null hypothesis is that the exogenous DNA transferred from the sperm to the blastocyst cells does not become integrated with the host genome and is destroyed by intrinsic DNAse enzymatic activity. The objective was to determine the fate of the internalized exogenous DNA in mouse blastocysts after brief or continuous exposure to DNA carrier sperm in vitro. The cytoplasmic and nuclear components of the cells were sepaMATERIALS AND METHODS

Polymerase chain reaction enzyme-linked immunosorbent assay detection of mycoplasma consensus gene in sperm with low oocyte penetration capacity**Presented at the 52nd Annual Meeting of the American Society for Reproductive Medicine, Boston, Massachusetts, November 2 to 7, 1996.

OBJECTIVE To determine the prevalence of mycoplasmas in washed sperm and to compare the penetration of zona-free hamster oocytes by sperm with and without mycoplasmas. DESIGN Prospective comparative study. SETTING Clinical and academic research environment. PATIENT(S) Semen from 34 male patients. INTERVENTION(S) Specimens were divided, Percoll washed, and scanned for differences in kinematic parameters. Sperm DNA was extracted and assayed for mycoplasma DNA using the polymerase chain reaction-ELISA method targeting the consensus gene of 15 mycoplasma species. Remaining sperm were processed by centrifuge, Percoll, or TEST (TES and Tris) Yolk Buffer (TYB) and assessed for penetration capacity. MAIN OUTCOME MEASURE(S) Detection of mycoplasma DNA. RESULT(S) Mycoplasma DNA was detected in 29.4% of the Percoll-washed sperm. The penetration of oocytes by mycoplasma-positive sperm (59.5% +/- 17.3%; mean +/- SEM) was less than mycoplasma-negative sperm (86.8% +/- 5.4%) in the TYB-processed group. CONCLUSION(S) Mycoplasma DNA is demonstrated in almost a third of the Percoll-washed sperm. Because there were no other cell types except sperm, the results suggest that the mycoplasmas were either internalized or attached to the membranes. The reduced penetration by mycoplasma-positive sperm after 48-hour TYB suggest mycoplasmas required time to affect sperm function. Similarities between hypo-osmotic swelling and between kinematic parameters suggest that the mechanism does not involve differences in membrane integrity and in motility patterns.

Sperm as a noninvasive gene delivery system for preimplantation embryos**Presented at the Annual Meeting for the Society of Gynecologic Investigations, Chicago, Illinois, March 22 to 26, 1994.

OBJECTIVE To determine if sperm could be manipulated to be a noninvasive transport carrier for the delivery of gene fragments to the blastocyst. DESIGN Sperm cells carrying foreign DNA fragments from human papillomavirus (HPV) types 16, 18, 31, and 33 were allowed to migrate from one end of an artificial reproductive tube and to come in contact with hatching mouse blastocysts at the other end of the tube. The blastocysts were then washed and analyzed for the presence of the foreign DNA fragments. SETTING Clinical and academic research environment. MAIN OUTCOME MEASURE Detection of amplified products from transferred foreign DNA using the polymerase chain reaction and primers targeted at the E6-E7 region for different HPV types. RESULTS Polymerase chain reaction analyses showed transference of DNA HPV type 18 to the blastocysts. Not all types of DNA fragments were transferred equally. CONCLUSION The results suggested the possibility of using sperm as a noninvasive gene delivery system for passing on gene fragments to preimplantation embryos. It was demonstrated that certain DNA fragments were easier to deliver than others, indicating the necessity for exploring all the factors involved in the mechanism of the transference process. The study also serves to highlight the possibility of unintentional transmission of viral or bacterial DNA to the developing embryo via the sperm.