On Assignment for Brown Medicine Magazine at the Warren Alpert Medical School of Brown University in Rhode Island. Feature story on the translational solutions for treating male infertility. Subject matter experts: Mark Sigman, MD, Chief of Urology at The Miriam Hospitals and Krishnamurthi Family Professor of Urology, Professor of Medical Science Kim Boekelheide, MD, PhD, Kathleen Hwang, MD, assistant professor of surgery, and Edward Dere, PhD, a postdoctoral research associate. Sidebar article details Dr. Hwang’s research on male reproduction at the Scott Department of Urology at Baylor College of Medicine.
Read below or download/view PDF of issue. View pgs. 32-37.
The Origin Of Man
Basic and clinical researchers team up to find translational solutions for treating male infertility.
By Nikolas Charles
Winter 2013
In the beginning there is love. Unfortunately, for some couples, their DNA is not in sync with their feelings. When biological complications prevent couples from conceiving, they typically experience the gamut of emotions, from sadness to intense anxiety. At the same time that they cope with their pain, most couples try every- thing to find a successful resolution.
“We see couples all the time who can’t conceive,” says Mark Sigman, MD, chief of urology at Rhode Island and The Miriam Hospitals and Krishnamurthi Family Professor of Urology. “Once it’s been determined that it’s not a female problem, a semen analysis is ordered. If the semen analysis comes back abnormal, we decide if we can do anything to reverse the problem and make the man fertile again. If we can’t find a cause in order to determine a treatment, the only option is to do in vitro fertilization or intrauterine insemination.”
According to Professor of Medical Science Kim Boekelheide, MD, PhD, “About 15 percent of couples are infertile. Half of the time it’s because of a male factor problem, which means more than 7 percent of men aren’t producing sperm that have the capability to fertilize an egg.”
The problem is often spermatogenesis — the formation and development of sperm. So in addition to sending patients’ semen to the lab for analysis, Sigman recruits his patients to participate in a major study that, researchers hope, will determine the molecular causes of sperm abnormalities that hinder conception.
“Currently the semen analysis is the best diagnostic tool to assess spermatogenesis in men,” says Kathleen Hwang, MD, assistant professor of surgery (urology). Yet that analysis may come back normal in men who can’t conceive, she adds. Indeed, the measurements in a typical sperm analysis — including count, motility, morphology, semen pH, and volume — are relatively unreliable.
“The semen analysis can tell us that the semen is abnormal, but it can’t tell us why it’s abnormal.”
“The semen analysis can tell us that the semen is abnormal,” Sigman says, “but it can’t tell us why it’s abnormal. There could be a lot of different things causing an abnormality. We can’t tell by looking at the analysis what’s causing it or what we can do about it.”
To try to find what underlying molecular characteristics, or biomarkers, of sperm may be impacting male fertility, Sigman and Hwang began collecting semen samples from 200 to 400 patients for a study called the Human Biomarker Project. Researchers also consider genetic, lifestyle, and environmental causes as they try to determine why couples can’t conceive.
“Collecting samples from our clinical practice for research purposes gives us the opportunity to develop sperm molecular biomarkers that may more accurately reflect fertility,” Hwang says. “This will potentially allow us as clinicians to better evaluate and counsel patients for male factor infertility.”
Show Us A Sign
Boekelheide and Sigman created the Human Biomarker Project, which is funded by the Lifespan health care system and the Krishnamurthi endowment, to understand the molecular traits that may cause male infertility. “Identifying these biomarkers is a priority for anyone working on male infertility research,” says Hwang, who came to Brown in 2011 to do research and clinical work.
A biomarker is an indicator of a biological state that is measured to assess a certain condition. In this study, “that state could be decreases in sperm concentration and/or motility that lead to decreased fertility or infertility,” says Edward Dere, PhD, a postdoctoral research associate who earned his doctorate in biochemistry and molecular biology from Michigan State University. He joined the research team in 2010.
Dere analyzes the semen samples that Sigman and Hwang collect from patients for changes in the epigenome, the compounds that “tell” the genome what to do and where and when to do it; and sperm transcriptome, which is the complete set of RNA, including messenger RNA (mRNA) and microRNA (miRNA), in a cell. These factors will serve as more sensitive predictors of male infertility. That’s because the RNAs inside the sperm could contribute to its health and fertilization capability. Lifestyle factors, like smoking, alcohol consumption, body mass index, or prescription medication use also may alter the transcriptome and reduce sperm function.
“There are epidemiologic studies that suggest that smokers have reduced fertility,” Sigman says, “but we don’t have a good way of identifying it. Having a biomarker for the effects of smoking would be very useful.” He says epigenetics, or heritable changes in gene expression, are not easily identified by traditional parameters, making the molecular biomarkers invaluable.
It was the opportunity to work with Sigman and Boekelheide on the human aspect of the research that brought Dere to Brown. “I felt that by working with human samples, my research would have a more direct impact than if I only worked with animal models,” Dere says. “Because of the collaborative nature of our team, we’re able to readily obtain human samples and investigate interesting questions.”
Sigman says, “Ed has developed great expertise in these techniques and has really moved our projects forward by deter- mining the best approaches to utilize.”
Back in the lab, Dere purifies the sperm in the semen samples by lysing, or rupturing, the somatic cells and washing away the seminal fluid. This ensures that the molecular profiles obtained from the sample are solely from the sperm cells. Semen samples can have sperm counts ranging from a few million to hundreds of millions. After analyzing for DNA methylation (the attachment of methyl groups to the DNA, which affects gene expression) as well as the RNA con- tent, Dere looks at the traditional semen analysis for possible correlations.
“If we find that men with low sperm motility also had consistent changes in their DNA methylation profile, we would have a predictive biomarker,” Dere says.
Boekelheide and Dere also focus on sperm mRNA, which is delivered to the oocyte, or egg cell, and may serve an important function in fertilization or embryogenesis. For this purpose the researchers use transcriptomic assays, an analytic procedure that allows for the simultaneous measurement of levels of all the genes in the genome, as well as polymerase chain reaction (PCR)-based arrays, which offer gene expression analysis. These lab techniques allow Dere to focus on a subset of genes that are identified from the sperm transcriptome profile.
“These projects are very high tech,” Sigman says. “They involve techniques that couldn’t be done a decade ago. Now we’re able to look at thousands of genes and changes in gene expression on little computer chips.”
“These projects are very high tech. They involve techniques that couldn’t be done a decade ago.”
The research also involves a great deal of biostatistical analysis. “With the methylation studies, one single experi- ment on one patient would generate possibly 480,000 data points,” Dere says. “If you multiply that by 400 patients, that’s a lot of data points. That’s where statistical analysis and computational techniques really come into play.”
Sigman doesn’t yet know whether biomarkers will replace semen analysis or turn out to be supplemental. “It would give us an understanding of the cause of infertility that we didn’t have before,” he says. “Ideally, it would allow us to ask the questions: Is there a signifi- cant male problem, and if so, what might be causing it?”
The Human Biomarker Project has the potential to address two lines of inquiry: Sigman’s research on male infertility and Boekelheide’s interest in testicular toxicity, including medications that can harm the testicle — a concern of pharmaceutical companies. “We’re hoping that the basic science experiments could help us identify certain patterns that indicate there’s testicular injury,” Sigman says. “From there, we might learn what kinds of things cause injuries, including environmental toxicants and medications. Knowing what the risk factors are and if there’s anything that we can do about it would help me clinically as I treat patients. Kim’s expertise has been a valuable resource for this type of translational research.”
Boekelheide leads several research initiatives on testicular damage caused by toxicants, including an animal model involving bisphenol A exposure. BPA, a chemical found in common plastic products, is the subject of a national consortium study to determine the severity of its effects. The team is also gathering preliminary data to submit a grant for a human model subproject to the Human Biomarker Project on how bariatric surgery may affect sperm biomarkers.
Bench to Bedside
Sigman came to Brown to study male reproduction and infertility in both a research and clinical setting. After be- coming chief of urology in 2009, one of his main goals was to grow the research arm of the division.
Although Sigman and Boekelheide had been collaborating for nearly 20 years, they wanted to develop a more robust research initiative. “Our history goes back a long way,” Boekelheide says of his relationship with the veteran clinician and expert in male infertility. “When Mark came to Rhode Island Hospital and Brown University in 1989, as a practicing urologist, he already had a strong background and interest in research from a urologic perspective.”
“Our goal is to develop a self-sustaining research machine that can continue this translational research.”
Sigman first did research in the area of immunologic male infertility during his residency at the University of Virginia. His work led to a project for the FBI’s Department of Forensics, where he developed antibodies to detect sperm in crime scenes. “That’s what got me interested in the field [of male infertility],” Sigman says. “I also wanted to continue doing research along with clinical care. After that, I did a two-year fellowship in male reproductive medicine and surgery at Baylor College of Medicine in Houston, just doing male infertility.”
Boekelheide became interested in male infertility research during his residency at Duke University. After graduating with an MD/PhD in anatomic and clinical pathology, he joined Brown’s faculty in 1984 to focus on research and teaching.
He and Sigman are combining their strengths, Boekelheide says: “I bring a basic research lab that has focused on animal models of testicular injury and male reproductive health effects using animal models. Mark brings a clinical practice in urology and his interest in male factor infertility.”
Since 2000, they have published four papers together on various topics, from basic research in Boekelheide’s lab to
the clinical translational challenges that Sigman faces as a practicing urologist. “We think of ourselves as a model of how to develop the research capacity of Brown and its affiliated hospitals,” Sigman says.
The pair believes Brown could replicate that model across other subspecialty areas. “I think the big picture is how we put the pieces together across the various institutions that make up Brown medicine,” Boekelheide says. “That has allowed us to do basic to clinical translational research in an effective way.”
Through basic research and applied clinical practice, Sigman, Boekelheide, Hwang, Dere, and the other scientists working on the Human Biomarker Project are making strides to help couples start their families. And, incidentally, they may be building a model that the entire University can copy for future, groundbreaking research projects.
“The people involved, both at Lifespan and Brown, have been very support- ive and like the concept,” Sigman says. “Our goal is to develop a self-sustaining research machine that can continue to pursue this kind of translational research.”
SIDEBAR
Scar Tactics
A chance discovery may help prevent problematic adhesions that form after surgery.
By Nikolas Charles
In addition to her efforts on the Human Biomarker Project, Kathleen Hwang, MD, is working on a project with a seemingly broader scope. The assistant professor of surgery (urology) brought the research initiative to Brown in 2011 from her fellowship in male reproductive medicine and surgery at the Scott Department of Urology at Baylor College of Medicine. The study investigates the role of ghrelin, a peptide that is produced in the stomach, in wound healing and in post-operative inflammatory response. Her findings could help to prevent scarring associated with testicular and abdominal surgery.
“It’s a phenomenal translational project, looking at a tremendous clinical problem,” says Mark Sigman, MD.
For now, Hwang is using an animal model to try to answer her clinical questions, which are based on her operating-
room experience with adhesions, or scars. “Her original impetus to go down that path was because she was working on infertility-related surgery and adhesions that arose there,” says Kim Boekelheide, MD, PhD.
room experience with adhesions, or scars. “Her original impetus to go down that path was because she was working on infertility-related surgery and adhesions that arose there,” says Kim Boekelheide, MD, PhD.
Hwang says she “stumbled upon” the project during her fellowship in Houston. “I was looking at ghrelin to see if it would prevent damage or interference with the sperm production process in an undescended testicle (cryptorchidism) model in rodents,” she says. “Then I discovered that the mice that were treated with ghrelin, compared to the control treated mice, had almost no post-surgical adhesions.
“Post-surgical adhesions are the bane of the surgeon’s existence,” Hwang continues. Any time a surgeon opens any kind of tissue, scars will form; in the abdomen, those adhesions form to the bowels, she says. Patients requiring repeated surgeries may suffer inadvertent injuries to their bowel due to these scars, necessitating further treatment.
"The application of the peptide ghrelin could reduce post-surgical scarring after vasectomy reversal."
“When I stumbled across this,” she says, “I thought, ‘If we found something that could actually prevent post-operative
scarring, that could change the way we operate and how patients can heal.’”
scarring, that could change the way we operate and how patients can heal.’”
The connection between surgical adhesions and urology (let alone male infertility) wasn’t immediately apparent, but
Hwang, who estimates that she dedicates 40 percent of her time to this project, ultimately connected the dots.
Hwang, who estimates that she dedicates 40 percent of her time to this project, ultimately connected the dots.
“One of the things that Mark Sigman and I do as ‘fertility people’ are vasectomies and vasectomy reversals,” Hwang
says. “The failure rate with vasectomy reversals is caused with the new anastomosis,” or rejoining of the vas deferens. During this part of the surgery, “a scar tends to form very quickly,” she says. If this happens, the lumen, which is the inside of the vasdeferens or epididymis, the tubular part of the spermatic duct system, can become blocked, preventing the sperm’s release from the testes.
says. “The failure rate with vasectomy reversals is caused with the new anastomosis,” or rejoining of the vas deferens. During this part of the surgery, “a scar tends to form very quickly,” she says. If this happens, the lumen, which is the inside of the vasdeferens or epididymis, the tubular part of the spermatic duct system, can become blocked, preventing the sperm’s release from the testes.
Hwang is now investigating how ghrelin affects wound healing and whether an application of ghrelin to the vas
deferens during the anastomosis can indeed minimize scar formation. “That would potentially increase the success rates of pregnancy in couples who still can’t conceive because of problems with scarring after a vasectomy reversal,” she says. “Our goal would be to help couples conceive naturally.”
deferens during the anastomosis can indeed minimize scar formation. “That would potentially increase the success rates of pregnancy in couples who still can’t conceive because of problems with scarring after a vasectomy reversal,” she says. “Our goal would be to help couples conceive naturally.”