Safeguarding the Foreigner Within

RMs are fairly common. On average one in five pregnancies will end in miscarriage and some research shows that there are up to 800,000 miscarriages a year in the United States.

Some causes are known, such as chromosomal abnormalities in the fetus, or hormonal imbalance, infection, or lifestyle choices that include the mother drinking, smoking, or taking drugs. But in the majority of cases in which genetic, anatomic, endocrine, and infectious causes can’t be proven, women are left wondering what went wrong.

Many researchers suspect that the mother’s immune response actually accounts for as many as 80% of unexplained cases. Fetuses express paternal antigens early in development, which triggers the immune systems of some mothers to attack the embryo as foreign, just as it would a bacterium or virus. Pregnancy requires tolerance of these foreign fetal antigens encoded by paternal genes.

When I started working in this field almost 9 years ago, I had high hopes that I could one day tell my great grandchildren that I had worked on the cure for miscarriage. I never imagined that in such a short time, I would have found a single drug candidate with the potential to prevent pregnancy loss.

In December of 2000, I came to the Hospital for Special Surgery, Weill Medical College of Cornell University, from Universidad Nacional de Rosario in Argentina where I was working on immune-mediated diseases in the kidney. When I saw an opening in the area of pregnancy complications, I jumped at the opportunity to work on a research question which I felt connected to personally. I received a career development award from the Lupus Foundation to study the cross-talk between inflammation and pregnancy loss, and quickly got up to speed in the field. I wrote to one of the researchers, Hector Molina from Washington University in St. Louis, whose work on pregnancy loss I had admired. He invited me to spend a week in his lab learning how to dissect embryos and perform the relevant assays.

By the 1950s, researchers had identified a class of antibodies that react against the placenta of a growing fetus called antiphospholipid antibodies (aPL). This class of antibodies binds to phospholipids, a major component of the cell membrane. About 30% of women who miscarry have elevated levels of aPL antibodies. Women today are regularly tested for the presence of aPL antibodies. But in 2000, there was no clear understanding of how these antibodies came into play in miscarriage.

For years, researchers had assumed that aPL antibodies caused fetal death by inducing thrombosis (blood clots) in placental blood vessels and cutting off blood flow and oxygen to the fetus. aPL antibodies had also been associated with stroke, thus it was assumed that since blood clots cause stroke, they must also be the cause of fetal death and miscarriage in pregnant women. It is the reason that women diagnosed with increased aPL antibodies are treated with anticoagulants or blood thinners such as heparin and/or aspirin throughout their pregnancy. The treatment, however, doesn’t always improve outcomes and presents the mother with risks, including a higher chance of uncontrolled bleeding during birth and surgery.

Since the relationship between miscarriage and aPL antibodies was only correlative, our group decided to develop a mouse model of aPL-induced pregnancy loss to understand the mechanism behind the correlation. I injected mice between days 8 and 12 of pregnancy (comparable to a woman’s second trimester) with aPL antibodies derived either from patients with high levels of aPL antibodies or mouse monoclonal aPL antibodies produced in the lab. Passive administration of any of these aPL antibodies caused 40% of the embryos to die.¹ When we looked at the tissue surrounding the embryos there was no evidence of blood clots. However, we did see that the dead embryos and the placentas were covered with antibodies, complement split products, and inflammatory cells, which we identified as neutrophils.² Antiphospholipids were known to bind specifically to a plasma protein called ß2-glycoprotein I (ß2GPI), which is expressed in high quantities on trophoblasts—a major cellular component of the developing embryo, but no one had thought that the complement system might be involved in miscarriage.

Complement proteins and receptors are crucial molecules in immune defense. Increased complement activation is usually observed after an organ transplant rejection. Since the fetus, with its foreign tissue, is like an organ transplant, it made sense that complement activation might be involved in fetal rejection or miscarriage. After an antibody binds a pathogen—or any other target—complement proteins from the blood rain down around it, ultimately forming a pore in the membrane of the pathogen and damaging it. While we didn’t see pore formation on the trophoblasts, another component of the complement cascade, C5a, was attracting and activating large numbers of neutrophils. By depleting neutrophils, or blocking the receptor for the C5a with antibodies, we were able to prevent embryo loss in the mice. The neutrophils were releasing toxic oxidative substances that injured the placenta and ultimately caused the rejection of the fetus. We published these findings in 2003.²

These were the first studies to underscore the importance of inflammation rather than thrombosis in fetal injury associated with aPL antibodies. I wondered whether heparin—used to prevent recurrent miscarriage—might actually function as a complement inhibitor during pregnancy. Others had shown that in addition to inhibiting clotting factors, heparin also blocks the complement cascade.

This idea made sense since we noticed that the doses of heparin used to treat women with miscarriage were too low to prevent clotting. So we tested other anticoagulants that don’t inhibit complement activation and saw that these did nothing to protect the fetus; complement was clearly at the center of the aPL miscarriages.³

Despite growing evidence that inflammation was an important factor in miscarriage, many clinicians were still convinced that thrombosis was playing a role. It was true that aPL antibodies were capable of inducing blood clots in humans, so we started to research the mechanism of aPL-induced thrombosis. We found a paper in the literature⁴ showing that a molecule important in coagulation—the first step toward thrombosis—is upregulated in patients with elevated aPL. The molecule, tissue factor (TF), is crucial for initiating the coagulation cascade. TF is normally expressed on tissues such as those in the outer layer of the blood vessels, which are not exposed to blood. But when a cut puts blood in contact with cells expressing TF, they initiate clot formation to stop the bleeding. Recently, researchers had made the connection that TF not only plays a role in the coagulation system, it also has proinflammatory effects through interaction with protease activated receptors (PARs).

Might TF contribute to inflammatory fetal death? We went back to our aPL model and found increased TF staining surrounding the dying embryos. To check the result experimentally we inhibited TF with monoclonal antibodies and saw that indeed, pregnancies were rescued by TF inhibition.⁵

A number of inflammatory signals, including complement components, increase the expression of TF on different cell types: endothelial cells, monocytes, and neutrophils. In collaboration with Nigel Mackman’s lab at The University of North Carolina at Chapel Hill School of Medicine, we studied mice that lacked mouse TF and were manipulated genetically to express low levels of human TF. The mice were protected against aPL-induced pregnancy loss, because in the absence of TF, the neutrophils were not activated and did not harm the fetuses. The picture that began to emerge was that aPL antibodies bound to the placenta, initiating the complement cascade, which then upregulated TF expression in neutrophils. The TFs then interacted with PAR-2 which activated neutrophils, causing the release of a storm of oxidants and enzymes that damage the placenta (see graphic below).

Researchers had noted that statins—drugs prescribed widely to lower cholesterol —could also reduce TF synthesis, expression, and activity in many different cell types including blood monocytes, endothelial cells, and breast carcinoma cells.⁶ The next step seemed obvious: Try statins as a therapy in our mice.

When antiphospholipid (aPL) antibodies bind to the developing placenta, they activate the complement cascade (1). One of the complement proteins, C5a, binds to the C5a receptor (2) on neutrophils, which upregulates the expression (3) of tissue factor (TF). Tissue factor interacts with the protease activated receptor 2 (PAR-2) to activate the neutrophil, which then releases reactive oxygen species and enzymes (4) which damage placental cells. Statins downregulate both TF and PAR-2, blocking neutrophil activation and preventing pregnancy loss.

It was quite an exciting prospect. Statins can be administered orally and would be much safer for women than heparin. In Phase IV trials of statins performed by Merck, women reported taking the cholesterol-lowering drug throughout their pregnancy with no adverse events. I started to plan the experiments, taking every care to ensure the experiments were private until we could publish. Everywhere I referred to the statin treatment—in the labels on mouse cages and in my notebooks—we called it “Substance Z,” shorthand for Zocor, the Merck-produced statin. On the binder that contained my study protocols and results, I pasted a sticker of the Secret Squirrel—a Hanna-Barbera cartoon character from the 1960s that sported a trench coat and a hat pulled over his face with two holes for his eyes. It was a tense but exciting time.

To test whether statins were downregulating TF in neutrophils and whether they could prevent pregnancy loss, we injected statins a day before injecting the mice with aPL antibody. To our delight, pregnancies were protected from miscarriage.⁷ The statins diminished both TF and PAR-2 expression, reducing inflammation while keeping the coagulation cascade intact. I soon started planning clinical trials for application in women.

With these successes under my belt, I set out to determine the other causes of miscarriage with an immunological basis. aPL-induced pregnancy loss accounts for 25–30% of immunologically mediated miscarriages. We decided to test whether TF might also be involved in the remaining majority of cases. We started with a model of miscarriage that didn’t require aPL induction, but in which complement played a role. In this model, pregnancy loss is caused by the inability to properly form blood vessels. In response to C5a generation, monocytes and macrophages infiltrate the uterus, releasing antiangiogenic factors that sequester VEGF—a potent angiogenic cytokine—and prevent the formation of the placental and embryonic blood vessels. We found that the TF expressed by monocytes and macrophages is required for the release of the antiangiogenic factor that kept the placenta from forming properly. We were able to demonstrate that statins, acting on TF, also prevented pregnancy loss in this model.⁸

These results were exciting for two reasons. They suggested an overarching immunological mechanism that could be behind the majority of immune-induced miscarriages, a mechanism with a drug candidate that was already approved and available generically. Logistically, it also meant that we would be able to expand the number of participants in our clinical trial. Our plan is to administer statins to women who have had more than three miscarriages, with and without aPL antibodies.

For me all of this excitement has come with a measure of disappointment. For political reasons and lack of funding, I have had to leave the Hospital for Special Surgery at the end of September.

On the bright side, I have teamed up with Michael Paidas and Charlie Lockwood, who are doctors of gynecology at Yale University School of Medicine and a number of other clinical collaborators. Together we are looking for funding to test our mouse findings in humans.

It’s frustrating to come so close to a finding that could benefit thousands of women, only to be thwarted by seemingly administrative issues. I imagine that women reading this story will feel as if I’ve left them with a cliffhanger. I hope that the next installment brings a clear resolution to the question of statins and miscarriage. For now, though, I regret to leave readers with a “to be continued….”

Guillermina Girardi was formerly an assistant professor at the Hospital for Special Surgery at Weill Cornell Medical Center in New York.