When it comes to reproductive fertilization, the process of natural selection is visibly at work. Thousands and thousands of tiny sperm struggle to penetrate the egg. They scurry and burrow into the egg’s membrane in a competition of strength and virility until eventually, only the strongest, healthiest sperm wins and breaks through.

For many couples, this is the simple biological process that results in conception of a child. Since on average, a male emits 20-200 million sperm per ejaculation, the odds are good that at least one sperm will be motile enough to complete the fertilization task. If the male has a particularly low sperm count, however, his chances for fertilization are dramatically reduced. And in cases of sterilization or certain abnormalities in which there is no sperm produced at all, the chances for conception were completely absent--until now.

Researchers and physicians at the Wayne State University School of Medicine have not yet found a way to treat this type of male infertility, but they have found a way to overcome it with a new process called intracytoplasmic sperm injection, or ICSI. Through this revolutionary new advance in reproductive technology, doctors need only one single sperm to fertilize an egg. In addition, the sperm doesn’t have to be mature or particularly active to make its own way to the egg. In fact, all that is required in most cases is a tiny sample of tissue from the testes where the immature forms of sperm are contained.

Pioneering this micromanipulation effort to increase fertility rates for would-be parents is Dmitri Dozortsev, MD, PhD, assistant professor of obstetrics and gynecology at the WSU School of Medicine. Dr. Dozortsev has performed hundreds of ICSI procedures in which he injects a single sperm into an egg using a specially-designed microscope and micromanipulator. The success rates have been astounding for couples who previously had no fertility options left.

"Female infertility accounts for about one-third of infertility problems, but we’ve made lots of progress in treating it," said Dr. Dozortsev. "When it comes to male complications, on the other hand, we’ve have had no real advances in the last decade. We learned how to induce ovulation in the woman, and we could extract eggs to make the process easier in a laboratory setting, but still--we had to rely on a strong sperm to fertilize the egg on its own. If it couldn’t fight its way through the zona pellucida--the outer shell of the egg--we couldn’t do much to help it. The only other option was to use donor sperm, but that wasn’t always an option couples wanted to pursue."

Using a specially designed microscope and micromanipulator, Dr. Dozortsev injects the sperm into the egg.

Thanks to ICSI and the advances it has introduced, doctors and researchers can now give the sperm a necessary boost through direct injection into the egg. "The genetics are there," said Dr. Dozortsev. "We just do what it takes to bring them together."

The Wayne State University department of ob/gyn, clinically based at Hutzel Hospital, has been bringing these genetic elements together for more than a decade. In fact, Wayne State developed the first in vitro fertilization program in Michigan in 1983, and more recently established a human genetics program to focus on research, diagnostics and genetic therapies. The vast growth of WSU’s micromanipulation techniques has increased the potential success for couples using in vitro fertilization (IVF).

In traditional IVF, fertility drugs are given to the female partner to stimulate the production of multiple eggs. After the eggs are surgically removed, the healthiest one is chosen to be placed in a petrie dish with approximately 250,000 - 500,000 sperm, said Michael Diamond, MD, director of reproductive endocrinology and the IVF program at WSU and The Detroit Medical Center. "We cross our fingers and wait for a sperm to bind with the egg. Even though we place the two gametes together, the process still requires certain cellular events to transpire for the sperm to penetrate the oocyte, or egg. Those cellular events may not occur if there are male infertility factors such as low sperm counts or poor movement."

Dr. Diamond explains that ICSI involves a simple one-to-one transfer of genetic material, whereby a single sperm is picked up by a special tool created by Wayne State ob/gyn researchers who found other tools too big and awkward for these microscopic manipulations. The tiny injection needle has a sharp or beveled end which makes it easier to pierce the egg membrane.

	The sperm is temporarily immobilized and loaded into a tiny needle.
	It then takes approximately one minute to inject the sperm through the membrane into the center of the egg.
	Once the needle is removed, the egg closes and resumes its normal shape.

The sperm is temporarily immobilized in a drop of solution so that it can be loaded into a pipette. It then takes only about one minute to inject the sperm through the membrane into the center of the egg. Once the needle is removed, the egg membrane closes and resumes its normal shape within a minute. After about eight hours, fertilization can usually be confirmed by the appearance of two pronuclei which contain the chromosomal and genetic materials from each parent. The egg is then implanted back into the female within 72 hours and the pregnancy progresses.

While in Belgium, Dr. Dozortsev was part of the original team that perfected ICSI and he directed one of the world’s largest ICSI programs between 1993 and 1996. Before that, he worked as a post-doctorate fellow at the Reproductive Genetics Institute in Chicago, IL, where he acquired expertise in assisted fertilization, micromanipulation and assisted hatching methods. He earned his medical degree and doctoral degree in Leningrad, USSR, where he became fascinated with embryology and histology.

"Fifteen years ago, these concepts were all brand new," said Dr. Dozortsev. "We are advancing our knowledge at such a rapid rate now, that I see endless possibilities for positive genetic therapies."

 

Make something of nothing

ICSI is a great alternative for men with very low sperm counts; but even when no sperm is produced, there are ways to extract sperm cells for fertilization, said Dr. Dozortsev. Generally, some tissue is taken from the testes and then it is examined for sperm cells, or the immature forms of sperm. This can be a tedious process because they are packed within tubules, and may be difficult to find.

Dr Diamond explanes medical advances that allow WSU doctors to perform pre-implantation testing for IVF candidates to screen for genetic disorders.

The critical element for success in the ICSI process is the sperm associated oocyte-activating factor (SAOAF). Since SAOAF is present in all living spermatozoa and spermatogenetic precursors in humans, all living spermatozoa are potentially able to fertilize an egg.

In fact, researchers say that in the future, there will be no need at all to distinguish between healthy and unhealthy sperm. They say that dead sperm and pre-cursors to sperm will do just fine. They still contain the relevant DNA materials to fertilize the egg and produce a healthy child.

Researchers extract a cell from this primitive eight-cell embryo for genetic testing.

 

Genetic testing - why wait?

For couples who get pregnant by natural methods, genetic testing is done through amniocentesis or chorionic villus sampling when the fetus reaches a certain stage of maturity--usually at least nine weeks. For couples who get pregnant through IVF assistance, there’s no need to wait. The eight-cell embryo that forms within 2-3 days of fertilization can be tested before it is even implanted into the womb. In fact, if a serious genetic defect is uncovered, the couple can choose to simply do ICSI again until they are able to implant a healthy embryo. This also eliminates the difficult decision of whether or not to terminate a pregnancy if a serious problem is found.

This earlier-than ever testing effort is being led by Dr. Dozortsev and Mark Hughes, MD, PhD, who have continued to advance Wayne State’s reputation as the premier site for reproductive genetic therapy. "This technology is primarily designed to assist high-risk mothers," said Dr. Dozortsev, "particularly IVF candidates who are at advanced reproductive age, are potential carriers for a genetic disease, or have a family history of genetic abnormalities."

The actual genetic diagnosis, a highly specialized molecular biology technique, is done by Dr. Hughes, who was recently recruited to Wayne State from the National Institutes of Health. "Dr. Hughes is not one of the best researchers in this field," said Dr. Dozortsev. "He is the best."

Through highly complex molecular techniques, Dr. Dozortsev removes a single cell from the very primitive eight-cell fertilized egg. Then Dr. Hughes examines the cell by mixing its DNA (containing the genes from the parents) with reactive agents. He amplifies the single gene of concern into millions of copies for subsequent genetic testing. The results are available in about eight hours, so that embryo transfer can occur the next morning.

"It is like finding a typographical error of one letter in a genetic book as large as 300 volume-sets of the Encyclopedia Britannica," said Dr. Hughes. "And we need to do this with our ‘molecular spell-checker’ on a microscopic cell in just a few hours."

For the most part, if a genetically inherited condition can be diagnosed in the prenatal period, it can also be detected in the preimplantation period. These detectable conditions include some forms of cystic fibrosis, hemophilia, Down’s syndrome, Duchenne muscular dystrophy, Fragile X syndrome and many others which are far more rare. "Preimplantation diagnosis makes it possible for parents with serious inherited disorders to decrease the risk of having a child affected with the same disorder," said Dr. Diamond. "There are a surprising number of couples who’ve suffered through repeated unsuccessful pregnancies due to genetic disorders. Many of them come to us with no hope left. But as our techniques and tools get more advanced, we’re replacing that hopelessness with healthy deliveries."

Currently, Wayne State University is the only site in Michigan offering preimplantation diagnosis to couples, and Dr. Diamond believes the possibilities for future advancements are limitless. "Our new molecular-level understandings are allowing us to help more people than ever before. It is at this level that we can do a lot of good."

 

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Wayne Medicine 1999

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