Immunofluorescence may improve microdissection testicular sperm extraction outcomes

San Francisco-Injecting an immunofluorescent dye into the testes may eventually help clinicians locate individual sperm and improve the yield from microdissection testicular sperm extraction (mTESE), researchers from the University of Iowa, Iowa City, reported at the American Society for Reproductive Medicine annual meeting.

But it's too early to suggest immunofluorescence-assisted mTESE for infertile patients. All of the experiments to date were performed in mice-and current protocols include removing entire testicles for microdissection.

About half of the six million U.S. couples who are infertile have at least one component of male infertility. Thirty percent have pure male factor infertility, Dr. Greenhalgh said.

mTESE has been used successfully in cases of nonobstructive azoospermia, but there can be significant difficulty in visually identifying areas that contain sperm within the testes. mTESE has a 40% to 50% sperm retrieval rate, which makes any technique that can increase sperm retrieval rate a potential improvement over current practice.

"We hoped that this immunofluorescence technique would help improve the procedure," Dr. Greenhalgh said.

The idea is that tagging individual sperm with fluorescent markers could help clinicians identify foci of sperm during mTESE. Identifying sperm foci could boost sperm retrieval for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).

Testing the concept

Preliminary in vitro testing using a fluorescent dye (fluorescein isothiocyanate, or FITC) conjugated with mouse anti-Human acrosomal IgM antibody (HS-14) produced easily visible sperm. The dye bound readily with both the head and tail of previously extracted mouse sperm, producing bright, glowing sperm that were easy to identify.

A group of 13 fertile mice and eight sterile mice that had been genetically engineered for early spermatogenic arrest were anesthetized and the testes exposed. Researchers used a microsurgical knife to make a small incision near the tunica albuginea near the vascular pedicle to expose the seminiferous tubules. HS-14 plus a small amount of crystal violet powder was injected into the exposed seminiferous tubules. After the dye was injected, the testicles were removed for microscopic examination.

Sperm were easily visible in all 13 of the fertile mice, Dr. Greenhalgh reported. Fluorescent dye bound to both the head and the tail of sperm in 22 of the 26 testes. It was particularly easy to visualize fluorescing motile sperm, he added, but even immobile sperm were readily visible. No sperm were seen in any of the 16 testes from the sterile mice. Histopathologic review of the sterile mice testes also failed to show the presence of any sperm.

Whether immunofluorescent-labeled sperm are viable for IVF or ICSI is still unknown, Dr. Greenhalgh added. There are no sperm-specific toxicity data on HS-14 or the antibody binding process itself and no data on the effects of binding or unbinding the fluorescent label on sperm viability, motility, and other properties. There are also no data on potential effects of immunofluorescent labeling on sperm DNA or a resulting embryo.

"Immunofluorescence may be the next step for improving sperm retrieval rates in microdissection mTESE," he said, "but it isn't as easy as it looks. There are some major engineering and biological questions to be addressed."