ONGOING RESEARCH | Dr. Sweet, Fertility, IVF

Introduction:
The use of cryopreserved sperm is common in many Assisted Reproductive Technology (ART) procedures. Some men with very poor quality semen have their semen frozen prior to In Vitro Fertilization (IVF) to assure an abundance of sperm is available for fertilization. Cancer patients frequently rely on semen cryopreservation as a means to preserve their fertility just prior to surgery, radiation and chemotherapy. Cryopreserved donor sperm from “sperm banks” is available for patients who have no sperm. One of the side effects of freezing sperm is that much of it does not survive the freeze/thaw process. In most instances, this is not a tremendous issue because literally millions of sperm are still alive after the thaw. In some cases, however, the number of motile sperm is low and a very significant portion of the sperm doesn’t survive the thawing process.

An ongoing study at SRMS involves the use of Hyaluronic Acid (HA) to increase the survival rate in thawed sperm. The procedure is accomplished by incubating the sperm in media containing variable concentrations of HA prior to freezing. By hopefully increasing the number of sperm that survive the thawing process, the number of quality sperm available for various ART procedures may be increased which should theoretically increase the chances of fertilization and a positive pregnancy test.

Abstract:
Increased Post Thaw Survival Rates Observed In Semen Specimens Treated With Hyaluronic Acid

Andrew Bhatnager, Ph.D., Corey Burke, B.S., C.L.S., Anna Hackenberg, Craig R. Sweet, M.D. (10/2008)
Hyaluronic acid (HA) is a naturally occurring mammalian glycosaminoglycan. It is a found in the cumulus-oocyte complex and seems to augment fertilization itself. HA has also been proven to be beneficial for in vitro mammalian embryo development with an increased the post-thaw survival of cryopreserved embryos. It is involved in numerous dynamic processes including regulation of gene expression, cell proliferation and cellular differentiation.
It was hypothesized that the addition of varying concentrations of HA would increase the post-thaw survival rate as well as the percentage of rapidly progressing human sperm.

A dose response curve was first established to determine the most effective concentration of HA to be used in semen preparation and freezing media. Specimens were obtained from men presenting for routine semen analysis at an infertility clinic. Only those specimens with normal motility (>50%) and progression (a-b) were used in the study. The specimens first underwent a gradient separation technique followed by a single wash in modified Human Tubal Fluid (mHTF). The motility and progression values were obtained using a Computerized Assisted Sperm Analyzer (CASA). The overlying supernatant was removed and the pelleted sperm were divided into three equal portions. One portion was resuspended in mHTF (control), a second was re-suspended in mHTF containing 0.001 g/ml of HA while the third was re-suspended in mHTF containing 0.025 g/ml of HA. All samples were incubated at 37¬æC for one hour and then centrifuged. Equal parts of Test Yolk Buffer containing the same original concentration of HA were added. The specimens were then frozen in liquid nitrogen using a controlled slow freeze method.

Specimens were later thawed at room temperature and washed in HA-free mHTF. Post-thaw results were obtained following the wash and repeated at 1 hour later using the computer assisted semen analysis (CASA).
In specimens incubated with 0.001 g/mL HA, the overall post-thaw motility was 45.5% with 18.7% of the sperm found to be rapidly moving. The sperm in the control group (no HA added) had a reduced motility of 37.5% with 16.5% rapidly moving sperm. Preliminary results, therefore, reveal an increase in survival and progression rates in those specimens that were frozen using a concentration of 0.001 g/ml of HA.

Dr. Sweet’s Comments:
It is common for fewer than half of the sperm will survive after thawing. Any technique that can improve the survival rate would be welcome. Increasing the function of the sperm through increased motility would also be ideal. In this preliminary study, HA was found to do just that. While more research certainly needs to be done, this is an important study that could have implications for a whole host of cryopreservation procedures.