25Wayne Medicine / 2002

AN INTERNATIONAL RESEARCH TEAM based atWayne State University is developing a simple, non-invasive screening test that will not only determinewhether a man is infertile, but will reveal the causeof that individual’s infertility. On a broader scale,the work may answer the widely publicized ques-tion of whether the general male population in theUnited States and other western nations is actuallyexperiencing a decline in fertility, and if so, identi-fy probable culprits as well as solutions.

For couples who are having difficulty starting afamily, the news means that men may soon be ableto skip the extensive battery of tests, including atesticular biopsy, that is currently part of the infer-tility screening. Instead, they may only need to pro-vide a sperm sample. In addition, the research teambelieves the test may provide enough specific infor-mation so that medical professionals can betterdirect treatments, or can identify which coupleswill never conceive. The latter diagnosis will allowcouples to forgo the lengthy, expensive, and ulti-mately futile infertility therapy, and begin consider-ing other options, such as sperm donors.

This promising new test will likely have a broadreach, remarked one of the team members.“Infertility involves about one out of every six cou-ples who are trying to conceive, so it is actually ahuge clinical problem that many couples face,” saidMichael Diamond, MD, the Kamran S. MoghissiProfessor and associate chairman of the WSUobstetrics and gynecology department. He addedthat most infertile men actually produce sperm, butfor some reason, those sperm are unable to fertilizean egg. “If this new technique does all of the thingswe’re hoping and expecting it to do, it could changethe way male-factor infertility is diagnosed aroundthe country and around the world.”

Beyond that, he added, “It has all sorts of poten-tial when you start looking at what effects pharma-ceutical drugs have on sperm, sperm function andother sperm characteristics. It can also possiblyestablish the effects of environmental toxins, alco-hol, caffeine, smoking or illicit drugs on sperm. Sothis work may have extensive impacts.”

Microarrays and mRNA Used in Male Fertility and Reproductive Health BY LESLIE MER TZ

Microarrays like this are helping to identify genes

associated with male infertility.

27Wayne Medicine / 200226 Wayne Medicine / 2002

Microarrays and mRNAWith their verification of mRNA in sperm and the addition of

microarrays to their research, the team’s work began in earnest. “Thefirst question we posed when the group got together was: Could weuse this technology to rapidly identify what mRNAs are present inhuman sperm?” Dr. Krawetz recalled. They could, and they did.

“We isolated some 20,000 mRNAs from a pool of normal individu-als just to get an idea of what the normal human population has,” hesaid. They then developed microarrays containing tiny sites that trapspecific mRNA. When sperm is added, color changes at each trap siteindicate whether the sperm includes each bit of mRNA. Almost imme-diately, the researchers can scan the sperm to tell which mRNA, andwhich associated genes, are present. They already have a basic profileof a fertile male, and are refining it now.

Next, they will begin to study infertile men. Using the same tech-nology, they will add infertile sperm to the microarrays to compare itagainst the fertile-male profile. They will then document the points ofdifference, run those discrepancies through an algorithm they aredeveloping with help from the WSU computer science department,and deduce the causes of male-factor infertility.

“We would ‘spot,’ or add, onto the microarray all the genes that wefind are associated with male factor infertility,” Dr. Miller described.“Then it would be a machine, basically, that would read off and givethe clinician some idea of a) what the underlying causes are, and b)what options the patient has for treatment or management of hisinfertility.”

Dr. Krawetz added, “On the basis of this work, we believe we willbe able to devise a preclinical screen for men or couples who are seek-ing infertility counseling. We should be able to detect a certain per-centage of the population who are infertile based on their patterns ofmRNA.”

Environmental HealthThis work may also have ramifications for male reproductive health

in general, the research team asserts. “We feel this is going to be quiteimportant in the next few years in not just unraveling what causes aman’s infertility, but also in helping us to understand whether therereally are reductions in sperm counts occurring in men in the West. Ifthat’s the case, we might get some handle on what’s causing that too,”Miller said, explaining that the normal-male profile will again providethe basis for the comparison.

“Over the last 10 years, there has been an increasing amount ofconcern about the potential effects of environmental exposures onhuman reproductive health,” reported team member David Dix, PhD,a research biologist and microarray specialist at the EPA. He cited therecent media coverage of endocrine-disrupting chemicals and theirpossible connection to male fertility as one example.

With the new normal-male profile and microarrays, Dr. Dix said,“We now have a window into the male reproductive system in whichwe can monitor overall reproductive health with precision. In fact, wecan get tens of thousands of different questions answered from a sin-gle experiment — not only predicting for us whether this individual

will be fertile or not, but giving us a detaileddescription of the gene-environment interaction forthat individual.” In the latter case, microarrayswould target genetic differences between the nor-mal-male model and men who have been exposed tosuspected toxins. “You can create a whole series oftools, or perhaps one very large microarray tool,that would allow you to monitor the potential effectsof environmental chemicals on male reproductivehealth,” Dr. Dix said.

The microarray test would give a man a quickdetermination of whether his sperm had beenadversely affected by a toxin. “That approach wouldtake into account not only how most people respondand the exposure limits that have been set on howmost people seem to respond, but how each indi-vidual responds,” he said, pointing out that differ-ent men have different genetic susceptibilities.

It would be particularly beneficial to men whoare at high risk of environmental-toxin exposurethrough the workplace, Dr. Krawetz added. A com-pany, for instance, could use the microarray test tomonitor its male employees for overall exposure-induced changes in fertility, while an employee whowas trying to start a family might request a test toensure he was fertile. If a problem did arise, theemployee could curtail his exposure, and simplywait the average 60-90 days for his body to replacethe old sperm with new, unexposed sperm. Dr.Krawetz noted, “This is the future of testing andpatient care.”

More Than Just DNAThe test grew from a finding made simultane-

ously in two labs an ocean apart. Stephen Krawetzat Wayne State and David Miller at the University ofLeeds in the U.K. separately verified that malesperm conferred something other than DNA duringfertilization. These male reproductive cells also car-ried mRNA, or the genetic message with the blue-print for making the sperm. Upon learning of hisresearch, Krawetz contacted Miller, and the twostarted working together. Miller soon decided tospend time in Krawetz’s lab, and is currently atWayne State pursuing their investigation.

“We thought that if we could analyze that mes-sage in the mRNA, and compare an infertile manwith men who are normally fertile — those who havefathered children — and see differences, then wewould be getting closer to narrowing down what thecause of that man’s infertility is,” said Miller, PhD,associate professor in the department of obstetricsand gynecology at Leeds. “In other words, we feelthat the mRNA message is crucial to understandingwhat’s gone wrong.”

The collaboration didn’t end there. “At the sametime, I met David Dix of the EnvironmentalProtection Agency,” said Krawetz, PhD, of the WSUobstetrics and gynecology department, and the uni-versity’s Center for Molecular Medicine andGenetics (CMMG) and Institute for ScientificComputing. “We put together this research trianglebetween Wayne State, the EPA and the University ofLeeds, and through that, I was invited to be one ofthe founding members of the EPA MicroarrayConsortium (http://www.epa.gov/nheerl/epamac).This partnership enabled us to produce arrays thatwere capable of examining expression of testes-spe-cific genes, which allowed this research to advancemore quickly than relying on commercially avail-able arrays,” Dr. Krawetz said.

Microarrays have become a crucial component ofthe project because of their capabilities as high-techindicators that can quickly search out specificgenes present in genetic material, and detectwhether they are affecting fertility.

Patents and PotentialIn addition, their research may be a boon to ani-

mal husbandry, he continued. “Beef production is agood example. Cattle have a long gestation time, soif you could ensure that every pregnancy outcomewas successful, you could increase your beef pro-duction, and the profit margin would then go upconsiderably.”

Dr. Miller added, “It can be applied to any animalof economic value to help improve the breedingstock. We know that most other animals that havebeen studied have mRNA in their sperm, too, so itwouldn’t be difficult to extend this to examine thespermatozoa of those animals.”

Buoyed by their success and the great potentialof this work, the researchers have jointly filed pro-visional-patent applications on the technology, andanticipate full patents by the end of 2002.

Mostly, however, they look forward to theresearch yet to come.

“This shows that international collaboration isessential to furthering successful science,” Dr.Miller remarked. “It also shows that there is roomstill for communication beyond the bounds of theUnited States and vice versa, and it is going to befor the common good.”

Added Dr. Diamond, “We’ve heard so muchabout the Human Genome Project. At least on theOB/GYN side, this will be one of the originalapproaches that actually takes some of that infor-mation about all of the genes that have been identi-fied and brings it to the practice of medicine. Thisis the type of thing that can come from these sortsof investments in the future.” �

Dr. Krawetz is creating genetic profiles for fertile and infertile men.