Tales of Two Thrombophilias

Although we strive to practice “evidence-based medicine” when the evidence is good and the practice is sensible, there are many times when personal experience still plays a role in patient care activities. Nowhere does this seem to be more true in obstetrics than in situations where “thromophilias” potentially place a pregnant woman at risk or might be a contributing factor in recurrent pregnancy loss. Under those circumstances we must carefully weigh the risks and benefits of therapy vs. no therapy, and we must include in those equations a broader perspective, the emotional turmoil of pregnancy loss or the potential loss of a mother who has recently given birth to a child. The comments from the two readers below (my apologies to them for the modifications for the sake of clarity) and my responses may help put these thoughts into a better perspective, because my answers many not be what either expected, but they illustrate the various factors that must be considered when confronted by these sorts of issues.

• At Tue Nov 20, 12:48:00 PM 2007, Amber said…
I have factor V Leiden (heterozygote) and had one normal pregnancy without knowing that I had it. With my current pregnancy the doctors started me on Lovenox. My original doctor said that I would be switched from Lovenox at 36 weeks and induced at about 38 weeks. Because I am military, I don't really have the luxury of seeing the same doctor because of duty station changes, etc. At my most recent appointment, the doctor I spoke with said they weren't going to change me to heparin until 38 weeks and that they would not induce early. My son was 3 weeks early, so if this pregnancy chances to follow suit, shouldn't they change me to the heparin at 36 weeks like my original doctor advised? And is being induced 2 weeks early normal? I tend to believe my original doctor because the other one changes his mind constantly about even the littlest things. Thank you…

• At Mon Nov 26, 06:58:00 PM 2007, Kenneth F. Trofatter, Jr., MD, PhD said…
To Amber Nov 20: Sorry for the delay in responding but I just got your comment in my mailbox today. I need to have the answer to one VERY important question, before I can answer any of yours properly. What led to you finding out that you were a factor V Leiden heterozygote? Those tests usually don't get done on any routine basis! Anyway, personally, I would switch you to heparin at 36 weeks, especially if you delivered early before (and why was that delivery early? - another important question!) because if there is nothing else I have learned in this business, it is that obstetrical history tends to repeat itself. The reason for switching you is to decrease your risk of bleeding and anesthetic complications (bleeding at the site of an epidural or spinal) around the time of delivery. Most anesthesiologists would prefer that Lovenox be out of your system for 48-72 hours before you have a regional block performed. Heparin can be reversed easily with protamine sulfate, even if you present in labor just having taken a dose, whereas Lovenox cannot be reliably. Let me know the answers and I will see if I have any other thoughts. Thanks for reading! Dr T

To which Amber responded...

• At Tue Nov 27, 12:36:00 PM 2007, amber said…
I found out because my grandmother has had clots her entire life, and was finally tested a couple years ago. She had clots after all of her pregnancies and was found to be a factor V Leiden heterozygote. My uncle had a pulmonary embolism in ‘91, and so he tested as well, and was positive. My mother has had some minor clots, and tested positive for it too and told me about it. She said it was genetic, so I requested to be tested because I had been on birth control and I have heard that hormonal birth control can add to the risk. I haven't had any problems with clotting thus far, it probably helps that I am military and usually fairly active. I was not aware of this family history until after my first child. My son was early naturally and I didn’t have any complications with him thankfully.

• At Thu Nov 29, 11:05:00 AM 2007, Kenneth F. Trofatter, Jr., MD, PhD said…
To Amber Nov 27: Thanks for your answers. I figured there was more to the story! My first answer stands. Based on your family history and your previous early delivery, I would switch you to heparin at 36 weeks. However, if the baby is growing well, has normal fluid, normal nonstress tests, and normal Doppler flow studies, and you are not having any complications yourself, I would not deliver you before 39 weeks unless I first documented fetal lung maturity with an amniocentesis. This is the current recommendation of the American College of Obstetricians and Gynecologists. Late preterm delivery is now the number one cause of premature delivery and there is an increased risk of morbidity for both you and the baby if induced early when there is no good medical indication. After delivery, I would place you on Lovenox, starting 12-24 hours after having the baby, continuing that until at least 6 weeks postpartum. After that, you might consider taking one baby aspirin (81 mg) per day for the rest of your life in view of your very strong family history! Good luck my friend and let us know how things turn out! Dr T

The second patient was diagnosed with a different “thrombophilia” as the result of a workup for recurrent miscarriages, but otherwise has no medical history, family history, or past obstetrical history that would appear to place her at “high risk” for complications, or even necessarily implicate her thrombophilia as a cause of her recurrent early pregnancy loss…

• At Sat Nov 24, 11:06:00 AM 2007, Anonymous said…
Dr. Trofatter,
I was just diagnosed with 2 copies of the MTHFR C677T
mutation (homozygous). I am 38 years old, have a healthy 2 year old boy and have had 3 miscarriages this year, all pregnancies with the same father. We are meeting with a genetic counselor next week. I am having several tests done and have gotten the MTHFR result so far. I miscarried at 8 wks, 11 wks, and 5 wks. No testing was done prior to the 3rd miscarriage. I have gotten pregnant each time I have tried, though my doctor is still going to test my estradial and FSH. Do you think this is neccessary?

Here is my question- I have done a ton of research and have a basic grasp of the blood disorder but am wondering what actually stops the baby from surviving? Is it always that there is a defect because of the lack of folate and vitamins B getting to the fetus? Or is it that there is blood clotting and the baby isn't getting what it needs? I also want to know, and I know the geneticist will tell us more once she has mine and my husband’s results, but should we NOT try for another? This year has been gut-wrenching and I selfishly want another child, not just for me but for my son. I always wanted more than one child but is it too risky??? I am on Vitamin B12, B6, 5 mg of folic acid and 81 mg of baby aspirin. I 'm assuming I will get my homocysteine levels checked before I get the green light to conceive? I don't want to wait much longer because I am 39 years old in March. Thank you for any and all information regarding these questions.

• At Thu Nov 29, 05:16:00 PM 2007, Kenneth F. Trofatter, Jr., MD, PhD said…
To Anonymous Nov 24: You were homozygous for C677T when you had your first child and you did just fine. The presence of this homozygous MTHFR polymorphism only modestly increases your “risk” so that should only be a minor consideration with regard to your decisions regarding another pregnancy. To answer your questions, these polymorphisms may put you at slightly greater risk for clotting problems, particularly if you have other risk factors, but they are probably not a major cause of recurrent early pregnancy loss, especially once you have started supplemental folic acid. That probably also reduces the risk of having a baby with a neural tube defect or congenital heart disease as well.

First let me review why folate metabolism and the methylenetetrahydrofolate reductase (MTHFR) gene are important. MTHFR is an enzyme that requires folic acid to convert homocysteine to methionine (an important amino acid) and when this does not occur, homocysteine can accumulate. As we discussed in a previous post, when this occurs in a developing embryo as the result of either folate deficiency or certain mutations in the MTHFR gene, this may have a ‘toxic’ effect, increasing the risk for neural tube defects and certain cardiovascular abnormalities. This same biochemical pathway is also essential for the production of a substance called S-adeneosyl methionine that is an essential intermediate in pathways that add methyl (CH3) groups to nucleic acids (DNA; RNA), proteins, neurotransmitters, and phospholipids, a process that plays an important regulatory role in the biological functions of each of these. The MTHFR C677T polymorphism is associated with a reduced efficiency of these metabolic pathways, but this deficiency can be overcome by loading up on folic acid as you have done.

But, all that said and done, I would wager that your primary problems are more 'age-related' than anything else. Fertility drops with age for reasons that are not completely understood. Part of the contribution may be the increased risk for having a chromosomally abnormal fetus; part may be other medical problems such as diabetes, obesity, thyroid disease, autoimmune disorders, hypercholesterolemia, and hypertension; part can be anatomical changes within the uterus that impair implantation, such as fibroids, endometriosis, or endometrial polyps; part is simply that your body's ability to hormonally support an early pregnancy might not quite be what it was when you had your son.

Ovulation induction and progesterone support may be all that you need, but I would suggest you find a Reproductive Endocrinologist (if you haven't already) since he/she will be in the best position to move you through a thorough evaluation and treatment regimen, even if that ends up being just empiric therapy. As you are well aware, at your age, efficient and experienced medical care is very important if you hope to have another baby. Good luck to you and thanks for reading. Dr T

Labels: factor V Leiden, MTHFR, recurrent pregnancy loss

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Thanks to Prudence, M.D. for GrandRounds 4.10!

Many thanks to Prudence, M.D. for the time and effort placed into putting together this week's Grand Rounds 4.10. And, thank you so much for including my post "Cytomegalovirus (CMV) Reprise" written in response to a reader's query regarding the association of CMV with recurrent pregnancy loss (RPL). CMV probably does not play much of a role in RPL, but it is still the most common comgenital viral infection and the leading cause of chorioretinitis and sensorineural hearing loss in chidren - and most women have never heard of it or even realize that they have the virus and could pass it along to their babies in utero!

Labels: CMV, cytomegalovirus, recurrent pregnancy loss

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Pre-op Dr T!

Well, in about one hour and forty-five minutes I will become a ‘patient’ myself. That’s because at midnight, I can no longer eat or drink anything until after I have had my left shoulder operated on in the morning. I am NOT looking forward to that, but I also cannot stand putting up with the aggravation of waking up in the middle of the night screaming in pain (my wife really appreciates that) and not being able to reach behind and tuck my pants in properly without catching myself in the middle with a gut-wrenching ‘spasm.' It’s the ‘surprise’ attacks that get me the worst when I reach or bump into something in just the wrong way or the fetal shoulder dystocia hurts me worse than it does the baby. Anyway, I am not telling you all this for sympathy (but I will take your thoughts!). I am telling you all this because I got about 25 comments in my email box today stretching back a week or so, and I may be a little slow to respond over the next few days.

I am actually a lousy patient myself. I had a stretch of about 20 years when I didn’t go for a doctor’s visit. I also don’t know how I have done this to myself, but the same thing came up about 5 years ago on the right side as well. When the insurance company asked my wife if this was a “work-related injury” she laughed and told the interviewer that “if you consider operating on one too many 400+ pound pregnant woman over the years a 'work-related injury', then it probably is.” The interviewer didn’t know whether to laugh too or move on…she finally decided to laugh. After she thinks about it though, she'll probably try to squeeze my employer for 'work-related' benefits so the insurance company can back out of the expense of the procedure. OMG, orthopedists charge a heckuva lot more for 1-2 hour procedures than I ever imagined possible.

I am kind of scared about the surgery, not so much for the pain afterwards, but because of the pain MEDICATION and anesthetic side-effects. I don’t do well with either. I could never become addicted to narcotics because they all make me so nauseated that even the thought of taking some starts my stomach churning. Fortunately, I respond VERY well to ibuprofen. And, if that doesn’t work, there’s always Johnnie Walker (I didn’t say that!).

Anyway, time to go to bed and try to get some rest before 0500 rolls around. Really looking forward to my Hibiclens shower in the morning! I will probably start to go through caffeine withdrawal about halfway through the operation. Guess I should warn my orthopedist and anesthesiologist about that. I wonder if they have a patch for that, or maybe IV Starbucks Pumpkin Spice Latte – no, don’t hold the whipped cream! Talk to all of you again soon.
XOXOXOXOXOXOXXO
Dr T

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Late Preterm Birth - Patient and Provider Education is Still Needed

I have spent much of the last year preaching the gospel of “Thou shalt not deliver before 39 weeks, unless thou hast a good indication.” In general, the “word” has been well-received by providers, although many have voiced skepticism of its divine origin. Many were not aware of the short- and long-term morbidity of “Late Preterm” or “Near Term” deliveries and appreciated the information; many expressed concern about a “bad experience” and didn’t “want to take the chance” of not delivering a woman early for certain conditions; others were so tied to their patients or unhappy with their own “cross-coverage” that they routinely delivered patients on their schedules, “even if it is just a little bit early”; others were afraid of losing their patients to other providers; and, many were not aware of the recommendations of the American College of Obstetricians and Gynecologists decrying elective delivery before 39 weeks without medical indication or assessment of fetal lung maturity. Most physicians agreed that if they could get their local colleagues to stand firm on the issue, and resist “temptation” they too could see the light and adhere to the guidelines.

The primary concern expressed by the skeptics relates to the patient’s unacceptance of its truth. I was not entirely surprised by this observation because its prevalence is a daily reality in our own practice. The cacophony resounds in the valley of the shadows of perceptions: “I had my last baby at 36 weeks and it did just fine.” “My mother had me at 8 months and I turned out okay.” “I’m really starting to feel awful and believe you should take my baby now.” “If you won’t deliver me when I want to be delivered, then I will find another doctor who will.” “We can’t take our (15 year old) daughter’s whining any more, she’s suffering so much, you must deliver her.” Basically, the same patient comments were reiterated at every place I have spoken.

It is clear if this campaign in the wilderness is to be successful, women must be educated about its importance and proactively question the ways of the recent past. Induction rates at many hospitials exceed 40% and I am aware of some that exceed 80%. It has been clearly established that inducing labor, or even worse, performing an elective cesarean section, before 39 weeks without medical indication is associated with more risks than benefits for both mothers and babies. So, if you are a pregnant woman, be aware of the following facts, and if you are a provider, please be sure your patients are aware of them as well:

• Inductions with an unfavorable cervix require more medical intervention, are usually more uncomfortable, and can be prolonged
• Induction increases the risk of having a cesarean section, particularly in women having their first babies and an unfavorable cervix
• Prolonged inductions increase the risk for infection, intrapartum and postpartum, and postpartum hemorrhage
• As a consequence of the above, there is an increased need for parenteral antibiotics and transfusion
• Babies born between 34-38 weeks have a greater risk for respiratory problems, hyperbilirubinemia, hypoglycemia, infection, thermal instability, feeding problems, prolonged hospitalizations, admissions to the neonatal intensive care unit, readmissions following discharge from the hospital, long-term morbidity and even death
• Babies risks are increased for all the above even further if delivered by cesarean section, especially if the cesarean is done in the absence of labor
• ALL the above can result in longer hospitalizations, risk for unexpected complications, and increase the cost of medical care

Remember, before there was exogenous pitocin (prostaglandins, laminaria, foley bulbs, and combinations of all), women often went into labor by themselves at “term!” Although we have not sorted out all the mechanisms of spontaneous labor, generally, at term labor is signaled and accompanied by fetal “maturity” with lungs, brain, liver, and immune system ready to face the cold cruel world. Often times, the mother’s cervix is also more “ripe” by that point as well. There is no higher liability risk to providers if uncomplicated patients are watched until the onset of labor at least up to 41 weeks and with careful monitoring, even longer.

Labels: induction of labor, Late preterm birth

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Letter to My Readers

Dear Readers,
A week or so back I received the comment below buried under one of my posts. It troubled me a lot, but it came at a good time, because it made me focus on some of the concerns I have had regarding my role on this site and your expectations. Please read it (and my response) and then I will explain myself…

• At Wed Nov 14, 11:06:00 AM 2007, JAY said…
Hi! I am a 17 year old girl. I recently got pregnant by my boyfriend and got up to being a month old. I didn’t tell my mother anything. I began to bleed heavily and get abdominal pain. I didn’t go to the doctor because I didn’t see a point telling my mother if I wasn’t going to have the baby. This ALL just happened about a week ago. I just stopped bleeding and I feel much better. Do you have any advice what should I do?? I know I need to go to the doctor to get checked out but I don’t want to tell my mother!

• At Fri Nov 16, 08:50:00 AM 2007, Kenneth F. Trofatter, Jr., MD, PhD said…
To Jay Nov 14: I cannot tell you what you should do regarding your mother, but sometimes, Moms are much more understanding than you might think under these circumstances. You might want to give her a chance. I do strongly recommend you see a provider who can follow-up to confirm that you are no longer pregnant, check your blood type and antibody screen, test you for sexually transmitted infections, and counsel you regarding contraception. You might not have thought of any of these things, but they are all very important for your current health and future childbearing. Oftentimes all of these services can be provided by your local health department. Good luck! Dr T

Now for my concerns. First of all, I want all of our readers to know that I have been much appreciative of your response to “Fruit of the Womb.” I never expected the number or the diversity of your comments and am flattered by the trust you have put in me. Quite honestly, I spend more time trying to answer your questions than I do on writing fresh posts! But, when I do respond, let me remind you again of the limitations of my answers. I can provide some thoughts, some information, some clarification of something you do not understand, some questions you might ask your providers, and maybe even a correct solution (given enough information) to your particular problem, but I cannot possibly understand your entire situation and I certainly CANNOT replace your own doctors (or your Moms or Dads) in providing all the answers for your care.

If you have an urgent problem or a clinical decision that has to be made in a timely fashion, this is not the place to seek help. The internet cannot take the place of real-time, face-to-face, and hands-on attention of a physician or other provider who knows you well. So, don’t be afraid to ask them your questions, the same ones you are asking me! As I tell all my patients, NO question is insignificant, although you may be surprised that the answer is often much more complicated than the question itself. If they tell you something and you do not understand, then ask them to explain it in a way that you can. Don’t ever leave your doctor’s office with big questions lingering in your heads, unless of course your doctor tells you up front that “I don’t know myself at this time.”

I am also getting to the point where I cannot answer all of your questions or I cannot respond to them in the time frame you might need. Please do not hold that against me. Many of you have very complicated medical histories and problems and even then I must frequently have more information before I can even venture a response. You are much more likely to get a response, and one in a timely fashion, if you have very specific questions in mind. Remember, this site is not set up in a way that we can easily have a running dialog with readers and, quite frankly, that would be difficult anyway since I write these posts in my free time and I have my own patient care and teaching responsibilities. I would also ask that you indulge me in one other way. Sometimes I reserve the right to hold off responding to your questions, so that I can organize my thoughts in a full regular post when it is clear that many other readers might benefit from my response. The primary purpose of this site is to provide as much information to as many people as possible!

Anyway, thanks to all of you for reading and for the feedback and questions you have raised in response to the posts. I hope you have gotten as much out of this blog as I have!
Dr T

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Plasminogen Activator Inhibitor-1 (PAI-1): Role in Adverse Pregnancy Outcome? - 6 - Treatment and Response Accompany Improved Outcomes

In the last several posts, we have presented support from the literature that links imbalances in the fibrinolytic system, as reflected in increased activity of PAI-1, or a genetic predisposition for the same, with adverse pregnancy outcome, both late and early in pregnancy. With regard to specific mechanisms contributing to recurrent pregnancy loss (RPL) in early pregnancy, we have reviewed evidence to support that aberrations of PAI-1 production could potentially have deleterious effects on ovulation, establishment and maintenance of the corpus luteum (which is essential for ‘progesterone support’ of early pregnancy), and early implantation/placentation of the embryo. In our final post (whew, finally!) on this subject, let’s look at data that would support the premise that down-regulation of PAI-1 production can be accomplished during pregnancy, or in anticipation of pregnancy, in ways that would be safe for both mother and baby, and might improve pregnancy success. At the outset, let me tell you that there is a plethora of information regarding ‘treatment’ of PAI-1 abnormalities under various clinical circumstances, but I would like to highlight only a few articles that pertain directly to pregnancy and RPL.

Bremer and colleagues in 1995 (Am J Obstet Gynecol 1995;172:986-91) performed a small study in which they “assessed the effects of a daily oral dose of 60 to 80 mg of aspirin from 12 weeks gestation until delivery on fibrinolytic variables before and after parturition…in 24 patients, eight receiving low-dose aspirin and 16 controls…The only maternal fibrinolytic variable affected…was plasminogen activator inhibitor activity, which showed a significant reduction before and after parturition of 40% and 70%, respectively, in low-dose aspirin users compared with controls.” None of these patients were reported to have significant pregnancy complications. Since aspirin is not known to have a direct effect on PAI-1 production or activity, it was concluded that the reduction in PAI activity is probably the result of inhibition of platelet reactivity.

In another study published the same year, Gris and colleagues (Thromb Haemost 1995;73:362-7) identified 30 women with a history of unexplained RPL and “an impaired fibrinolytic capacity.” Without identifying the specific reasons for their fibrinolytic imbalances, these women were randomized to begin prior to conception either low-molecular weight heparin (enoxaparin) 20 mg per day or a phenformin-like substance, moroxydine chloride, 1200 mg per day. After one month of treatment, if their fibrinolytic status normalized, therapy was continued for 6 months with the intention to continue treatment if they became pregnant; and, if their fibrinolytic status did not improve after one month, they were switched to the drug they did not receive the first time. The results were actually quite dramatic. With regard to normalization of fibrinolytic status, 20 out of 29 women responded to the first- or second-line enoxaparin treatment whereas did only 1 of 19 treated with moroxydine. Sixteen of 20 enoxaparin responders conceived compared to only 2 of 10 nonresponders (p = 0.002); and, 13 of 16 enoxaparin responder pregnancies resulted in live births compared to none of the 2 nonresponders (p = 0.02). This was compelling evidence that ‘anticoagulation therapy’ at subtherapeutic levels with a heparin compound might improve pregnancy outcome in women identified to have underlying hypofibrinolytic imbalances without even delving into the specific causes of these imbalances.

In 2000, Bick (Clin Appl Thromb Hemost 2000;6:115-25) reviewed the results of anticoagulation therapy in women with histories of RPL and no identifiable chromosomal, hormonal, or anatomical defects. Of the 160 women analyzed, 150 (94%) were found to have coagulation defects, and 38 were found to have more than one of the defects for which they were screened. Their mean age was 33 years and their mean number of miscarriages before referral was three. 149 women were treated preconceptionally with aspirin (81 mg/day) and, immediately following conception, were begun on unfractionated heparin 5000U every 12 hours, both of which were continued until delivery. The remarkable results of this study were that only 2 of the 149 women failed therapy and to have a live birth. This translates to a ‘success rate’ of 98%! In a subsequent report (Bick and Hoppensteadt, Clin Appl Thromb Hemost 2005;11:1-13), among 351 women with RPL who had no other identifiable cause, 322 (92%) were found to have coagulation abnormalities. Those with ‘thrombophilias’ were treated preconceptionally with aspirin (81 mg/day), to which was added following conception, unfractionated heparin (5000U/24 hr) in the first 120 patients, or the low molecular weight heparin, dalteparin (5000U/day), in the next 192 patients. (Patients with MTHFR polymorphisms were also treated with folic acid 5 mg /day and pyridoxine (vitamin B6) 50 mg/day). As the authors reported, “Only 2 of the thrombophilia patients suffered another miscarriage; all others had a normal term delivery” for an overall success rate of 94%.

Also in 2000, Glueck and colleagues (Fertil Steril 2000;74:396-7) presented a case report of a 32 year old woman with amenorrhea and infertility associated with polycystic ovary syndrome (PCOS) who had failure of 7 out of 10 IVF embryo transfers, 1 premature live birth, and two pregnancy losses at 8 and 17 weeks. She was obese, had high fasting serum insulin, androstenedione, and testosterone levels, and was also found to have a modest deficiency in protein S and the 4G4G polymorphism of PAI-1, accompanied by high PAI-1 activity. The combination of the protein S deficiency and the elevated PAI-1 characterized her as having “familial thrombophilia and hypofibrinolysis.” Although not overtly ‘diabetic’, she was begun on metformin (2.55 g/day) and a weight reduction program. Metformin is an oral drug used to treat type 2 diabetes. It improves blood sugar control by various mechanisms, decreasing glucose production by the liver, decreasing absorption of glucose in the gastrointestinal tract, and probably, most importantly, by increasing insulin sensitivity, accompanied by improved peripheral glucose uptake and utilization. Over the course of 4 months, her weight fell from 109 to 91.3 kg (16%), her insulin, androstenedione, and testosterone levels normalized, as did her PAI-1 activity levels.

As a follow-up to this case report, Glueck’s group (Fertil Steril 2001;75:46-52) reported preliminary results from an ongoing pilot study to determine whether metformin could reduce the rate of first trimester pregnancy loss in women with PCOS. They identified 19 women with PCOS who did not have overt diabetes and placed them on metformin (1.5-2.55 g/day) throughout pregnancy. Ten of the women had previously conceived but had miscarried 16 of their 22 pregnancies (73%). “While receiving metformin, these 10 women had 6 normal live births (60%), 1 spontaneous abortion (10%), and 3 normal ongoing pregnancies (30%)” all > 13 weeks. Up to the time of the report, among all 19 women receiving metformin, 9 (47%) had normal term live births, 2 (11%) had normal, but preterm births at 33 and 35 weeks, 6 (32%) had normal ongoing pregnancies beyond 1 weeks, 2 (10.5%) had first trimester miscarriages. No adverse maternal side-effects, nor birth defects were attributed to metformin in this small study. Most importantly, for purposes of our discussion here, “among women who received metformin before conception, reductions in insulin and plasminogen activator inhibitor activity were correlated (r = 0.65; P = .04).” Thus, metformin alone appeared to improve pregnancy outcome in a group of PCOS patients who had either had, or were at increased risk, for early pregnancy loss.

In a subsequent prospective study, Glueck and colleagues (Clin Appl Thromb Hemost 2004;10:323-34) evaluated the efficacy of combined therapy with metformin (1.5 to 2.55 g/day) and enoxaparin (60 mg/day) in women with PCOS and one or more previous early pregnancy losses, thrombophilia, and/or hypofibrinolysis. “Of the 24 women, 23 had 65 previous pregnancies…with 18 live births, 46 spontaneous abortions (71%), and one elective abortion.” Of these 23 women, seven had 3 or more consecutive losses, two had 2 consecutive losses, thirteen had 1 loss, and one woman had a live birth in a pregnancy complicated by HELLP syndrome. Compared to ‘controls’ with no history of adverse pregnancy outcome, the 24 women in this study had a higher frequency of the factor V Leiden mutation (17% vs.2%; P = 0.016), the PAI-1 4G4G polymorphism (46% vs 24%; P = 0.031), higher levels of the PAI-1 gene product and PAI-1 activity (33% vs 8%; P = 0.018), and a higher frequency of elevated factor VIII levels (22% vs 0%; P = 0.037). Of the 23 women who conceived on enoxaparin-metformin to date in the report, they had had 26 pregnancies (28 fetuses), with 20 live births, two ongoing pregnancies > 13 weeks, and 6 spontaneous early losses (21%), 3.4-fold lower than in their previous pregnancies. Again, no adverse maternal or fetal therapy effects were noted.

The articles cited above, have suggested that under various conditions associated with fibrinolytic imbalance and RPL, correction of the imbalance is at least a marker for, if not a direct contributor to, improved first trimester pregnancy success. None of these studies have really confirmed that improvement in pregnancy outcome could be directly correlated with reduction in PAI-1 activity. In the early 1980’s it became recognized that women with PCOS, who ovulated poorly or not at all, would sometimes benefit from partial removal (wedge resection) of their ovaries, or even complete removal of an ovary. This was frequently accompanied by spontaneous ovulation and a decrease in the male hormones (androgens) that can be produced in excess by the ovaries of women with PCOS. Various techniques were employed over the years to reduce the ovarian tissue mass that resulted in the hormonal imbalances accompanying PCOS, but in 1989, Daniell and Miller (Fertil Steril 1989;51:232) described a laparoscopic technique termed ‘ovarian drilling’ in which 4-20 hormone-producing follicles (cysts) on one or both ovaries were pierced and cauterized using laser or electrocautery techniques. This procedure resulted in a dramatic decrease in male hormone levels within days, spontaneous ovulation in 70-90% of women, and a 40-60% probability of pregnancy within a year. Palomba and colleagues (Fertil Steril 2005;84:761-5) performed a comparative study of women with PCOS and elevated PAI-1 levels who underwent ovarian drilling with or without treatment with metformin. Ovarian drilling alone did not reduce PAI-1 activity, whereas metformin administration did. Furthermore they found that a lack of decrease in PAI-1 activity was related to a high risk of miscarriage in those women who conceived following ovarian drilling. These findings suggest that fibrinolytic imbalance, characterized by elevated levels of PAI-1, is an independent risk factor for RPL.

In closing, let me return to one more study by Glueck and colleagues (Metabolism 2006:55:345-52) that we cited in an earlier post. We mentioned previously that results in this study of women with PCOS demonstrated that PAI-1 activity was independently and positively associated with risk for first trimester miscarriage and that “for each 5 IU/mL increment in PAI-1 activity, the risk of being in an adverse first-trimester miscarriage category increased.” What we did not mention before is that prospectively, women in this study were placed on metformin prior to conception and their subsequent pregnancy outcome was assessed and correlated with changes in PAI-1 activity. “From pretreatment to the last preconception visit on glucophage (metformin), in 30 women who subsequently had live births, PAI-1 activity fell 44%, but rose 19% in 23 (also metformin treated) women with first-trimester miscarriage (P = 0.03).” Furthermore, “in the 30 women with live birth pregnancies, median PAI-1 activity fell continuously through the first trimester…, whereas PAI-1 activity was either unchanged or rose in women with first-trimester miscarriage.” Therefore, not only is increased PAI-1 activity an independent risk factor for RPL, but failure of response to therapy, as reflected in lack of normalization of PAI-1 levels also appears to be as well.

At the outset of this series, I dedicated the work to one of our readers (IR) who has had recurrent miscarriages and asked me many months ago about my thoughts on the relationship of PAI-1 activity and RPL. During the course of reviewing the literature I have come to the conclusion that there certainly is a correlation between the two. Perhaps the most compelling evidence resides in the observations that ‘appropriate’ PAI-1 activity appears to be a part of normal ovulation and implantation/placentation in early pregnancy. It is easy to speculate how imbalances at these critical times could interfere with early pregnancy success, regardless of the underlying causes that led to these imbalances. It would appear that efforts to improve a ‘hypofibrinolytic’ state, reflected in women with RPL and increased PAI-1 activity, should be considered as part of any therapeutic regimen. Preconceptional weight reduction, if indicated, treatment with metformin, low-dose aspirin, supplemental folic acid and B-vitamins, ovulation induction with progesterone support, and prophylactic use of heparin or low-molecular weight heparin under these conditions are all options that can be employed and have a wide margin of safety for both mother and baby.

So, IR, I hope this helps. And, I really hope that sometime soon you conceive the baby you are destined to carry. I know you will be a great Mom!
Dr T

Labels: fibrinolysis, PAI-1, recurrent pregnancy loss

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Cytomegalovirus (CMV) Reprise

There is not a week that goes by in my practice that I do not have to address questions regarding cytomegalovirus (CMV) infections and pregnancy. Since we have also been discussing recurrent pregnancy loss in various contexts over the past year, I thought her questions were particularly timely and would be of interest to a good number of other readers. This is a good opportunity to reprise some of the information about CMV that we have provided in the past…

Anonymous has left a new comment on your post "Cytomegalovirus (CMV): Common and Confusing":

I have had reoccurring miscarriages and my OB did a TORCH blood test which showed my CMV levels were slightly elevated. I’m supposed to go back in two weeks for another test to recheck the levels. Could this virus have caused my miscarriages? Is there a period of time I should wait before trying to conceive again? If I do become pregnant again am I at risk of having a baby affected by CMV?

To Anonymous: All of your questions are answered at the very end of this post. You can jump to there if you would like or read on for more information…

The TORCH test looks for antibodies in your blood specific for TOxoplasmosis, Rubella (German measles), Cytomegalovirus, and Herpes simplex virus. It is not unusual to include a screen for syphilis and human immunodeficiency virus (HIV) at the same time. All of these can cause infections of a baby during pregnancy (congenital infections) but by far, CMV is the most common. If antibodies to a specific organism(s) are present, this indicates you have been infected with that organism at some time in the past. Let me elaborate on that point a bit so that you have a better understanding of the tests when you discuss them with your doctor.

Usually, when we contract a virus infection like the flu, our immune systems react by first producing specific antibodies of the IgM class. These usually hang around only for no more than 2-4 weeks after the infection has been cleared. Shortly after IgM antibodies begin to be made, our bodies switch to the production of a second class of specific antibodies called IgG. IgG antibodies generally hang around for a long time after the infection is cleared and provide us with a source of 'permanent immunity' to the organism, helping to prevent reinfection, or decrease the severity of a secondary infection, with the same or similar organisms with which the antibodies might 'cross-react.' It is these IgG antibodies that also afford protective immunity to the baby because they can cross the placenta whereas IgM antibodies cannot.

We can use this information to help us to characterize the status of an infection. If neither IgM nor IgG is present, the individual has probably never been exposed to the organism of concern (or is too early in the course of the infection to have mounted any antibody response). If IgM is present and there is no IgG, then the infection is probably a 'primary' infection, indicating first time exposure to the organism, usually very early in its course. If both IgM and IgG are present, this also usually reflects a primary infection, but later in the course of the disease. And, if only IgG is present, then this indicates a state of permanent immunity established from an infection that occurred at some time in the past. In any of the first three circumstances, if an infection with a specific organism is suspected and could be of concern for a pregnancy, it is probably worth repeating the antibody titers in 4-6 weeks.

Things can be a little confusing with CMV infections. CMV grows very slowly and the incubation period from the time of exposure to onset of symptoms, or asymptomatic excretion of the virus, is on the order of 4-12 weeks. IgG antibodies usually can be detected by 1-2 weeks after the onset of symptomatic infections, but because of the long incubation period of CMV, this might be a month or more after actual exposure to the virus. The presence of IgM in the absence of IgG and in the presence of symptomatic disease (or a history of recent exposure to a known carrier) is highly presumptive of a true 'primary infection.' Similarly, the findings of both IgM and IgG with a significant rise (four-fold or more) in IgG titers (with or without a fall in IgM titers) on a follow-up screen 4-6 weeks later, usually (but not always in the case of CMV) indicates a recent primary infection. Also useful, the presence of IgG in the absence of IgM, is highly suggestive of a remote exposure to the virus, often greater than 6 months previously.

The rub with serology in classifying CMV infections comes in most often when IgM is present but IgG titers are relatively stable or mildly fluctuating. Unlike most common viral infections, CMV-specific IgM can sometimes persist 6-9 months following its appearance. And to make things even more confusing, IgM has been found to reappear on occasion with reactivation of latent CMV infections. In other words, except in the circumstances detailed above, we may not be able to use the presence of IgM in our counseling to tell patients that they have had a primary or recurrent infection during the pregnancy or if the infection might have occurred even prior to the current pregnancy.

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CMV is the MOST common congenitally (fetal) and perinatally (newborn) acquired virus disease in humans and the single most important infectious cause of mental retardation and congenital deafness in the U.S. and other industrialized nations. CMV is a member of the herpes family and human CMV is restricted to humans with no known animal reservoir. At least 80-90% of all individuals are infected with CMV during their lives, but infection may occur in the absence of clinical symptoms or without recognizing that an illness is the result of CMV. As with other herpes viruses, once an individual is infected with CMV, 'recurrences' can result from periodic reactivation of virus replication at various sites in which the virus is latently harbored in the body. During periods of reactivation, the virus can be more readily transmitted to individuals who have not been previously infected. Although probably not that common, secondary infections can also occur with other strains of CMV.

Transmission of CMV can occur from exposure to just about any body fluid, most commonly via saliva, respiratory, and venereal routes or by contact with infected urine or breast milk. Exposure tends to occur at earlier ages in lower socioeconomic groups, promiscuous individuals, and children at day care centers. Antibodies to CMV (seropositivity), indicating a history of infection with the virus, in women during the common childbearing years (18-35) can be found in about 50% of those in middle and upper, and 90% in lower, socioeconomic groups. Among previously noninfected (seronegative) women, the chance of becoming infected with the virus (seroconversion) is about 1-3% per year, however, this is as high as 10-20%in women who work in day care settings and 50% in women with infected children under two years of age!

Congenital infections with CMV occur in 1-2% of ALL pregnancies, accounting for 40-50,000 cases per year, indicating the virus can cross the placenta with relative ease compared to other herpes and most other common virus infections. Transmission of CMV to the fetus can occur with both primary and recurrent infections despite maternal immunity and has been documented in consecutive pregnancies. However, congenital infection rarely results in a poor outcome unless the congenital infection is the result of a primary (first time) infection in the mother during the pregnancy. Overall, among seronegative women, there is about a 0.5-1% risk of a primary CMV infection in each pregnancy, although this is much higher in 'at risk' groups such as day care workers, health care providers, and women with young children. Primary maternal infections carry an overall transmission risk to the baby of 25-50% and recurrent infections about 2-3%. Maternal antibody to CMV is incompletely protective against transmission to the baby, but it does play a major role in reducing the severity of infection in both fetus and newborn.

A high percentage of primary maternal infections are asymptomatic, or simply confused with another illness, or even written off as normal symptoms of pregnancy, so usually the diagnosis is not considered. Indeed, perhaps, the hardest part of making the diagnosis of CMV infection during pregnancy in mother and baby is simply suspecting that it might be a problem. If a woman develops a prolonged (weeks) illness with fever, resembling infectious mononucleosis (caused by another herpes virus, Epstein-Barr virus (EBV)), and the latter cannot be confirmed by routine testing, then CMV should be high on the differential diagnosis. The ante goes up considerably if the mother is a young teen having her first baby (especially if she conceived shortly after becoming sexually active or changing partners), or if she works, or has another young child, in a high risk setting such as a daycare center.

Serologic testing, as we discussed at the outset, can often help to confirm a primary infection when it is obtained coincident with maternal symptoms. Virus detection by culture, immunofluorescent, or polymerase chain reaction (PCR) techniques, best done by sampling maternal urine, may be positive (and almost always will be for months after a primary infection), but this alone does not tell us if this is a primary symptomatic CMV infection or a recurrent infection accompanying another illness. Unfortunately, in most cases maternal infection with CMV is usually not suspected until the baby is found to be growth restricted or has subtle physical abnormalities, or a thickened placenta, to suggest it has either a chromosomal abnormality or a congenital infection, sometimes months after the maternal exposure. Under these circumstances, maternal serologic testing might not be helpful in establishing either the fetal diagnosis or the diagnosis of a primary maternal infection during pregnancy.

Congenital CMV infections associated with primary maternal infections early in pregnancy and accompanied by growth restriction and detectable abnormalities by fetal ultrasound, generally, have a very poor prognosis, but even then, the outcome is not entirely predictable. Establishing the presence of fetal infection, when fetal abnormalities are identified, requires an 'invasive' procedure. Performing a simple amniocentesis, and using a CMV-specific PCR or shell vial culture technique, can confirm fetal infection with CMV in nearly 100% of cases. (Remember, the amniotic fluid from midtrimester on is mostly fetal urine, and CMV is excreted in large amounts from the kidneys following congenital infection, and often for years afterwards, even in 'asymptomatic' cases). However, unless we have confirmatory maternal serologic information, or symptomatic infection confirmed to be the result of CMV during the pregnancy, we still may not know if the congenital infection is the result of primary or recurrent maternal disease. If the diagnosis of CMV is not suspected until late in pregnancy, or not until after the birth of the baby, detection of CMV in a urine sample taken from the baby within the first two weeks'(preferably, the first 48-72 hours) of life also suffices to confirm congenital infection. CMV is a VERY slow growing virus, so any detection of virus in this time frame most certainly represents intrauterine infection.

Congenital CMV infections are asymptomatic or unrecognized in about 90% of cases, including 85-90% of babies acquiring the virus as a consequence of primary maternal infection and 99% of those resulting from recurrent infections. Most of the asymptomatic infections pose no immediate life threat, but 10-15% of these babies are at risk for long-term complications such as sensorineural hearing loss, chorioretinitis, and dental abnormalities, usually apparent by two years' of age. Of these, the hearing loss is by far the most significant because delay in its detection can contribute to psychomotor retardation. More than 90% of congenitally infected infants, regardless of the severity of their infection, will shed infectious virus at birth, and may do so for 6 or more years longer, even in the presence of specific immunity.

About 5-10% of congenitally infected babies will have significant evidence of infection at birth. Approximately half of these will have classic 'cytomegalic inclusion disease (CMID)' (described below) and half will have atypical involvement. Virtually 100% of these infants excrete CMV at birth and will continue to do so for many years afterwards. Infants in this group have a 20-30% eventual mortality and account for 90% of the significant mental and psychomotor retardation associated with congenital CMV infections. The most severely affected children are usually the result of primary maternal infections during pregnancy and should be suspected in instances of unexplained intrauterine growth restriction and fetal death beyond 20 weeks' gestation.

The most common findings in babies with CMID include symmetrical growth restriction, multiple small skin hemorrhages (petechiae), enlargement of the liver and spleen (hepatosplenomegaly), jaundice, microcephaly (small head), abnormalities of the dental enamel, and chorioretinitis. They can have numerous associated birth defects involving virtually any organ system, a catalog of which is not necessary for our discussion today. Prenatal detection by ultrasound of fetal growth delay and gross or subtle, nonspecific physical abnormalities of the baby may be the only clues that a congenital CMV infection has occurred. Unfortunately, these often manifest themselves only weeks or even months after fetal infection has occurred because the virus is very slow growing, and it is relatively nondestructive, compared to other herpes viruses.

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With the above information as background, let me answer our reader’s questions:

Could this virus have caused my miscarriages? It is unlikely. Although I did not address this specific point above, many people over many years have tried to implicate CMV in recurrent miscarriages and I have never seen an article that found good evidence to do so.

Is there a period of time I should wait before trying to conceive again? The TORCH screen and your CMV seropositivity should not preclude your getting pregnant again soon. You already have ‘protective immunity’ and you will probably have the virus for the rest of your life. However, if you have had recurring miscarriages, perhaps you should wait to conceive until you have had a more thorough evaluation for that.

If I do become pregnant again am I at risk of having a baby affected by CMV? Yes, you have a 2-3% chance of having a baby with a congenital CMV infection. But, the presence of your antibody to CMV will decrease the risk of serious complications from the infection. Most (99%) babies who get CMV as the result of a recurrent maternal infection are asymptomatic, but 10-15% of these babies are at risk for long-term complications such as sensorineural hearing loss, chorioretinitis, and dental abnormalities, usually apparent by two years' of age. Your baby can be screened within 48-72 hours of birth by simply checking his/her urine for CMV to determine if there was a congenital infection. Almost all babies with a congenital CMV infection will excrete the virus in their urine. If CMV is present, the baby simply needs to be followed more closely for the hearing and eye problems noted above to prevent correctible sources of developmental delay. These children usually do not have problems with growth or mental retardation.

Labels: CMV, cytomegalovirus, RPL

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Plasminogen Activator Inhibitor-1 (PAI-1): Role in Adverse Pregnancy Outcome? - 5 - Implantation and Placentation

Things have been so busy at work the last few weeks that it has been hard to find the time to write here, but I would like to finish up the series we started on plasminogen activator inhibitor-1 (PAI-1) and role in pregnancy outcome and recurrent pregnancy loss (RPL) before moving on to any other major topic. I will admit that the review of the literature on this topic has been somewhat tedious and at times confusing, but it has also been very educational from my own standpoint, and I figure, it’s okay to be a little selfish every once and awhile, especially if a few of our readers might also benefit from the results of the efforts. Anyway, in our last post we reviewed observations that have been made regarding the role of PAI-1 in ovulation and development (and regression) of the corpus luteum and posited possible deleterious effects on these events, under circumstances in which PAI-1 is elevated, that might lead to ovulatory defects and RPL in early pregnancy. Today we are going to turn our attention to observations that have been made regarding the role of PAI-1 in implantation and placentation.

During the first part of the menstrual cycle, prior to ovulation, the hormone that stimulates the growth and the development of the lining (endometrium) of the uterus is estrogen. As we discussed in our last post, following ovulation, the corpus luteum produces progesterone that prepares ('decidualizes') the endometrium to receive the fertilized egg; and, if implantation of the embryo occurs normally, the hormones (mostly hCG) that are produced by the developing placental tissues (trophoblast cells), stimulate the corpus luteum to continue to make progesterone until the placenta gets big enough to take over that function. In 1996, Lockwood and Schatz (J Soc Gynecol Investig 1996;3:159-65) reported that progesterone stimulation (decidualization) of estrogen-primed endometrial cells (estrogen up-regulates endometrial cell receptors for progesterone), both in vitro and in vivo, results in “a marked increase in the expression of tissue factor (TF) and type-1 plasminogen activator inhibitor (PAI-1) and an inhibition of tissue-type and urokinase-type plasminogen activators (tPA and uPA,respectively), matrix metalloproteinases (MMP), and endothelin-1 (ET-1) expression…” Thus, even prior to implantation, the fibrinolytic balance is shifted toward preservation of the extracellular matrix (ECM) structure and prevention of hemorrhage that could be imagined to occur as the embryo implants and the trophoblasts start eating their way into the maternal endometrium. Indeed, progesterone withdrawal shifts the fibrinolytic balance in the opposite direction and this is thought to be one of the factors that permits the shedding/bleeding from the endometrium with menstruation if pregnancy has not occurred successfully.

During the course of normal placentation, some trophoblasts invade the maternal endometrium to anchor the placenta, some migrate through the endometrium and then invade and remodel (open up) the maternal blood vessels (spiral arterioles) that will be the source of blood to the ‘placental bed’ from which the baby will extract oxygen and nutrients (and into which it will transfer its own waste products), and other trophoblasts develop into the frond-like villi that eventually dangle into the placental bed of maternal blood and actually perform these latter functions (transferring oxygen, nutrients, and wastes between the fetal and maternal circulations). Proliferating and invasive trophoblasts produce urokinase-like plasminogen activator (u-PA) and this helps to degrade the ECM and facilitates their migration within the endometrium. One of the primary roles of PAI-1 in normal placentation appears to be in controlling the degree to which trophoblastic cells actually invade the maternal tissues. Graham (Placenta 1997;18:137-43) found that a substance produced by maternal decidual cells, transforming growth factor-beta (TGF-beta), stimulates fetal trophoblasts to make both PAI-1 and the inhibitor of metalloproteinase-1 and also down-regulates trophoblast production of u-PA. This suggests that the trophoblasts, under the direction of maternal cells, may limit their own invasiveness by the secretion of these inhibitors that inactivate u-PA and prevent the degradation of the ECM.

This hypothesis is supported in a study by Floridan and colleagues (Placenta 2000;21:754-62). Beginning with the observation that “trophoblast invasion…in normal intrauterine pregnancies appears to be strictly regulated…whereas tubal and molar pregnancies seem to be characterized by uncontrolled excessive placental invasion,” PAI-1 localization was evaluated in both maternal and fetal tissues under these conditions. In normal pregnancies, PAI-1 was localized, predominantly to trophoblasts that were in closest proximity to maternal tissues: the basal plate of the placenta; the extravillous interstitial trophoblasts comprising the placental anchors in the endometrium; and the trophoblasts that replace maternal vascular endothelial cells as the result of the remodeling of the spiral arterioles. In the basal plate at the deepest layer of placental invasion, PAI-1 (secreted by the trophoblasts?) was noted to be associated with the surface membranes of maternal decidual cells “or confined to the extracellular matrix (ECM) facing the invasive front of anchoring villi.” In contrast, there was a paucity of PAI-1 expression by fetal trophoblasts and maternal cells in both tubal ectopic and molar pregnancies that accompanied uncontrolled trophoblast invasion and damage to maternal tissues.

So, if normal placental invasion of both the decidua and maternal spiral arterioles is at least partly the result of controlled and ‘normal’ expression of PAI-1 activity, we can easily speculate on the potential consequences of excessive PAI-1 production without defining the underlying cause. This could lead to very shallow invasion of the endometrium and inadequate migration to and invasion and remodeling of the spiral arterioles. If severe enough, this could result in early pregnancy loss and if less severe, could result in an abnormal placenta, typical of that seen in preeclampsia, accompanied by increased resistance to fetal and/or maternal perfusion and restriction of fetal growth.

There are many factors that might contribute to the increased expression of PAI-1 under these circumstances. In other posts we discussed the genetic polymorphisms such as 4G/4G that are associated with increased PAI-1 production and activity and this could possibly contribute from both the fetal and maternal sides. Since PAI-1 expression by trophoblasts is at least in part influenced by TGF-beta production by maternal decidual cells, then anything that contributed to increased TGF-beta production by either decidual cells, or cellular components of the maternal immune system, might also result in the fetal trophoblasts producing excessive PAI-1. Indeed, one could imagine that if an abnormal maternal immune response to the pregnancy, either innate or specific, was accompanied by the production of factors that up-regulated PAI-1 expression, this could also contribute to inhibition of trophoblast migration and invasion.

Personally, I believe that aberrations in the maternal immune response are a major cause of abnormalities of placentation that result in pregnancy loss and preeclampsia and there is some evidence indicating that modulation of PAI-1 expression is indeed one mechanism by which these deleterious affects are mediated. Bauer and colleagues (J Clin Endocrinol Metab 2004;89:812-22) reported that the cytokine, tumor necrosis factor (TNF) alpha, inhibits both invasion and migration of trophoblasts in tissue culture experiments. Furthermore, this inhibition is correlated with increased production of PAI-1 and can be reversed by specific antibodies against PAI-1, restoring normal trophoblast migration. In subsequent studies, Renaud and colleagues (Biol Reprod 2005;73:237-43) demonstrated that activated macrophages (components of the innate immune system) also inhibit trophoblast migration. They showed that this inhibition is clearly the result of TNF-alpha production by the macrophages, requiring specific binding of TNF-alpha to the trophoblasts, and that it is accompanied by decreased production of u-PA and increased production of PAI-1 by the trophoblasts themselves.

In our posts to this point, we have built the case that aberrations in the fibrinolytic balance, associated with increased production of PAI-1, accompany adverse pregnancy outcome both late in gestation and in early pregnancy, and have presented several mechanisms by which early pregnancy success might be impaired by increased PAI-1 expression. The questions then remain: can PAI-1 expression be down-regulated in individuals with increased PAI-1 and/or a genetic predisposition for the same and, if so, is this accompanied by improved pregnancy outcome? In our final installment in this series, we will present evidence that it can be and does!

Labels: PAI-1, placental abnormalities; spiral arterioles, recurrent pregnancy loss, RPL

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Grand Rounds 4.8 at Dr Anonymous!

Thanks to Dr. Anonymous for including my last minute submission regarding the role of endometriosis in recurrent early pregnancy loss (RPL)in this week's Grand Rounds 4.8. Many women associate infertility with endometriosis, but are not aware of the connection with RPL as well, so it should be of general interest to a good number of readers who have had miscarriages. Check out Grand Rounds - the selection of topics will keep you entertained for the next week!

Labels: endometriosis, grand rounds, recurrent pregnancy loss, RPL

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Plasminogen Activator Inhibitor-1 (PAI-1): Role in Adverse Pregnancy Outcome? - 4 - Ovulation and Corpus Luteum Development

In our last post on this topic, we concluded that imbalances of the fibrinolytic system are commonly found in women with no other obvious explanations for recurrent early pregnancy loss (RPL) and that one of the more consistent findings in this imbalance is elevated activity of plasminogen activator inhibitor-1 (PAI-1) and a genetic predisposition for the same. Today we are going to begin to review mechanisms by which increased PAI-1 activity could have a deleterious affect on early pregnancy success. In my search through the literature, I first came across several articles that addressed the role of PAI-1 and the fibrinolytic system in placentation. But, I also found several intriguing articles that addressed their roles in ovulation and in the development and regression of the corpus luteum. So let’s start our discussion at that point, since aberrations with ovulation and subsequent development of the corpus luteum might also increase the risk of RPL, and then we will move on to the observations that have been made related to placentation.

First, let me state that this is a very complex subject and in no way do I profess any specialized expertise in this area. Indeed, if any of our readers do have this expertise and want to set me straight or have another interpretation of the scientific literature, please feel free to comment. I am fully aware of my limitations and am here to learn too! Secondly, I am only going to focus on observations related to the fibrinolytic system. I am not going to address the plethora of factors that might affect the actual expression of the various fibrinolytic system components, plasminogen activators or activator inhibitors. So, I ask forgiveness for oversimplification at the outset…

During each normal menstrual cycle, ovarian follicles are ‘recruited’ to produce the ‘egg of the month’; these must undergo development and differentiation and usually one (and sometimes more) becomes the ‘dominant follicle’ and differentiates further. The follicle is comprised of an outer perimeter of extracellular matrix, theca, and granulosa cells that surround the egg and its accompanying (follicular) fluid. At the appropriate time in the cycle, under the influence of luteinizing hormone (LH), the extracellular matrix surrounding the follicle rapidly degrades, the cells at the leading edge of the follicle migrate to and penetrate the surface (capsule) of the ovary, and the egg and its immediate layers of surrounding cells detach from the inner layer of the follicular granulosa cells and is released from the ovary. Once free, the fallopian tube can embrace it, introduce it to friendly sperm along the way, and facilitate the ultimate journey of the fertilized egg to the uterus.

Following ovulation, what’s left of the follicle (the corpus luteum) begins to make the hormone progesterone that helps to prepare (decidualize) the lining of the uterus (the endometrium) to receive the fertilized egg, aiding attachment and implantation of the early embryo. There is a very narrow ‘implantation window.’ With implantation, the fetal cells begin to proliferate and invade the endometrium and they send hormonal messages back to the corpus luteum instructing it to remain healthy and to continue production of progesterone. If conception does not occur, or implantation fails too early, the corpus luteum degenerates, progesterone production drops, and the menstrual cycle starts all over again. So, where does the fibrinolytic system play a role in this sequence of events? Quite frankly, roles have been proposed for every step of this process, but let me focus on just a couple of areas of interest.

Remember from our first post on this subject, plasminogen activators (PA), such as tissue plasminogen activator (t-PA) and urokinase-like plasminogen activator (u-PA), convert plasminogen to its active form, plasmin, which can then direct the breakdown (fibrinolysis) of clots. Well, plasmin actually is in a class of compounds called serine proteases that can help breakdown and rearrange many protein compounds other than fibrin. With regard to the ovary (and other tissues), the extracellular matrix (ECM) is composed of a network of cross-linked protein compounds (mostly collagen) and ‘ground substance.’ In order for the follicle to develop, migrate to the surface of the ovary, and penetrate its surface, this ECM must be loosened up. Plasmin appears to play an important role in the remodeling of the ECM, thereby facilitating follicular cell migration, the actual rupture of the follicle and, perhaps, release of the egg from the follicle as well. PAI-1, which can inhibit both t-PA and u-PA (thereby limiting the production of plasmin), appears to play a key role in the regulation and balance of these events around and within the follicle and probably also plays a role in protecting less developed follicles from destruction as the result of plasmin and other proteolytic enzymes produced at the time of the LH surge.

In various animal studies and studies of tissue samples taken from humans in the periovulatory period, there is certainly evidence to support a role of the fibrinolytic system in the events culminating in ovulation and subsequent development (and regression) of the corpus luteum. Without belaboring this issue, I would simply like to make a few key points by citing a couple of articles on this complicated subject. Tsafriri and Reich (Exp Clin Endocrinol Diabetes 1999;107;1-11) reviewed the data to support the role of the fibrinolytic system in the degradation of the ECM. Following the LH surge, preeovulatory follicles are stimulated to make “a cascade of proteolytic enzymes, including plasminogen activator, plasmin, and matrix metalloproteinases (MMPs). These enzymes bring about the degradation of the perifollicular matrix and, most notably, the decomposition of the meshwork of collagen fibers which provides the strength to follicular wall. Pharmacological blockage of any of these enzymes resulted in the reduction of ovulation rate.” Instead of a “pharmacological blockade”, we could certainly imagine conditions wherein there is increased PAI-1 activity (reducing the activity of these proteolytic enzymes), inhibiting, or simply delaying ,ovulation and, thereby, disturbing the timing related to the ‘implantation window’ of endometrial receptivity and, perhaps, increasing the risk of implantation failure and early pregnancy loss.

Along the same lines, but dealing specifically with regard to the events surrounding follicular rupture and ovulation itself, several observations can be made. In granulosa cells from preovulatory follicles in humans, there appear to be very high concentrations of messenger RNA (mRNA) for both PAI-1 and PAI-2 and low concentrations for PA mRNA and this is reflected in a relative excess of PAI-1 over PA in the follicular fluid surrounding the egg (Jones, et al., J Clin Endocrinol Metab 1989;68:1039-45). In other words, until the egg is ready to hatch, inhibition of the proteolytic system predominates. Studies in the pig (Politis, et al., Biol Med 1990;43:636-42), however, indicate that immediately before ovulation occurs, PAI activity decreases and PA and plasmin activity increase, preceding ovulation. Thus we could imagine, just as we did with ECM degradation, under conditions of excess of PAI activity, ovulation might be prevented or delayed. To add support to this concept, excessive amounts of the pituitary hormone prolactin (PRL) are known to be associated with subfertility. Liu and colleagues (Hum Reprod 1997;12:2748-55) demonstrated in a rat model that PRL delays ovulation and this is accompanied by both a decrease in t-PA production and an increase in PAI-1 production.

Finally, let’s briefly mention the corpus luteum itself. The two primary plasminogen activators, t-PA and u-PA, appear to play divergent roles with regard to the CL. t-PA appears to be involved in the regression of the CL if pregnancy does not occur successfully, and its activity is directly correlated with a decrease in progesterone production. However, an “increase in u-PA mRNA and activity in the early stages of CL development is correlated with an increase in progesterone secretion” (Liu Biol Signals Recept 1999;8:160-77) and the development, growth, and support of the CL (Liu, et al., Endocrinology 2003;144:3611-17) that is essential for the implantation and survival of an early pregnancy. So, again we can use our imaginations. Since PAI-1 can inhibit both t-PA and u-PA, perhaps under circumstances of increased PAI-1 activity as seen in certain women with RPL, the balance shifts from u-PA predominance in the CL to t-PA predominance, causing the CL to regress rather than to grow to support an early pregnancy, thereby leading to pregnancy loss due to inadequate progesterone support. No data on this subject have I found…just a thought!

In our next post on this topic, we will (finally) look at the role of the fibrinolytic system in implantation and placentation….

Labels: corpus luteum, ovulation, PAI-1

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Endometriosis and Pregnancy Loss

Endometriosis is a condition that affects millions of reproductive age women. It is associated not only with infertility, pain, and other complications, but also with recurrent early pregnancy loss (RPL). Below is a series of comments and my responses to a woman with endometriosis and a recent cervical pregnancy. I hope this may provide some insight and information of general interest to women with RPL and endometriosis.

fryerfairy said...
I have endometriosis and had a cervical pregnancy. I was in the hospital for 3 weeks and I was treated with Methotrexate. I think it was 3 shots. I had this 6 months ago. I was told that I might not be able to carry a baby full term. Since having this 6 months on I am still in a lot of pain and am losing big clots at each period. I can no longer cope with the pain and the worry that I may never be a Mum I am only 23. Do you think I will go on to have a baby?
Sun Nov 04, 11:03:00 PM 2007

Kenneth F. Trofatter, Jr., MD, PhD said...
To fryerfairy: I wish I had my crystal ball for you. Let me ask a few questions: Did you have pain prior to the cervical pregnancy? Is this pain all the time or just at certain times of your menstrual cycle? Did they have to do any surgery in addition to the methotrexate? How do your doctors know that you have endometriosis? Have you had any treatment for your endometriosis to date? Are you having regular periods every month? Did you have any trouble conceiving the pregnancy that you lost? Do you have any other medical problems? Let me know and I will do the best to give you my thoughts. Dr T
Tue Nov 06, 12:18:00 PM 2007

fryerfairy said...
I have had 2 laparoscopies in which the endometriosis was first diagnosed and burnt away the second one I had they checked my tubes and ovaries and removed a lot of endometriosis from my pelvis. I had a miscarriage 6 months before falling pregnant again which resulted in the cervical pregnancy. I do suffer with a lot of pain and I can not remember the last time I was pain free. My doctor said that there is a lot of scarring and a lot of growths. Could this be the reason that the baby implanted in my cervix. I was due to begin IVF treatment as my husband and I have been trying for over 3 years when I fell pregnant. My cycles are regular but are odd and I do occasionally get spotting throughout the month. I don’t have any other medical problems. When I went to the hospital for an early scan they admitted me straight away but they did not know how to handle the situation due to it being so rare. I felt like a guinea pig having tests done
Tue Nov 06, 01:13:00 PM 2007

Kenneth F. Trofatter, Jr., MD, PhD said...
To fryerfairy Nov 6: By your history and symptoms, it could be that you have not only endometriosis, but ADENOMYOSIS as well. Adenomyosis is endometriosis in the muscle wall (myometrium) of the uterus itself. It can cause pain, anytime throughout your cycle, but especially with menstruation - if you can imagine bleeding into a muscle - by the irritation and inflammation that results. I am not an expert in Reproductive Endocrinology and Infertility (REI), but when very young women have severe endometriosis/adenomyosis they can have a great deal of difficulty conceiving and getting through first trimester and may have other underlying risk factors (autoimmune, hormonal, thrombophilic, and anatomical) that further increase their risk for recurrent early pregnancy loss. Some will have defects in the production of cell surface structures called 'integrins' as well. Integrins help the early embryo attach to and implant in the endometrium. Indeed, implantation defects seem to be a big problem in women with endometriosis that have recurrent early pregnancy losses (and, to answer one of your questions, in your case, perhaps, the embryo did not find a good place to implant until it reached your cervix).

Treatment varies but, it is my understanding, the first step is to surgically ablate as much of the endometriosis as possible. Many REI doctors will then place their patients on a course of Lupron (leuprolide acetate) for about 6 months to suppress the hormonal production that supports the endometriosis. This basically puts you into a reversible state of ‘menopause.’ (Yes, and I am afraid the symptoms of ‘going through the change of life’ are side-effects of the drug). You are a young woman and should consider this if you have not already had it. Then, controlled ovulation induction, sometimes with the use of Femara (letrozole – an aromatase inhibitor used to treat breast cancer and now found to be effective in ovulation induction and perhaps implantation under these circumstances), and even IVF may improve your chances for successful implantation and 'survival' through first trimester.

We used to say that getting through a pregnancy is the best 'cure' for endometriosis. I don't know if that still holds, but I have had many women with a history of endometriosis return within a year of a successful pregnancy having then conceived again spontaneously! In your case, one thing in closing...if you do succeed in getting through first trimester, because of the cervical pregnancy, you should be evaluated by ultrasound in second trimester to look for the possibility of cervical incompetence. Indeed, if there was significant damage done to your cervix by that pregnancy, your doctors might even consider placing a cerclage, prophylactically, at the end of first trimester. So, good luck and thanks for reading. Dr T
Wed Nov 07, 05:43:00 AM 2007

Labels: cervical pregnancy, endometriosis, recurrent pregnancy loss

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Plasminogen Activator Inhibitor-1 (PAI-1): Role in Adverse Pregnancy Outcome? - 3 - Association with Recurrent Pregnancy Loss

In our last post, we reviewed several studies that would lend support to the hypothesis that women with elevated levels of plasminogen activator inhibitor-1 (PAI-1), and a genetic predisposition for the same, are at increased risk for adverse pregnancy outcome later in pregnancy. Let me make it quite clear that these studies do not prove that increased PAI-1 causes preeclampsia (or any of the other conditions discussed), but they do suggest that PAI-1 may be a contributor to expression of the disease and particularly the more severe forms of preeclampsia. In today’s post, I would like to begin to build a case with the support of the published scientific literature over the years for an association of increased PAI-1 expression/activity (resulting in decreased fibrinolytic – clot breakdown - activity) with recurrent early pregnancy loss…

In 1993, Gris and colleagues (J Lab Clin Med 1993;122:606-15) evaluated the fibrinolytic system in 116 women who had recurrent early pregnancy loss (RPL) of unknown etiology matched with a group of 90 women who had never had an early miscarriage. Seventy-four of these 116 women with recurrent losses were found to have at least one abnormal test for fibrinolysis compared to none of the control group. A subgroup of 56 women who were shown to have decreased fibrinolytic activity in blood samples taken from veins that were intentionally occluded were selected for further evaluation. Seventeen of these women produced about half the amount of tissue plasminogen activator (t-PA) compared to the controls, 21 had elevated levels of PAI-1 activity, and 16 had both low t-PA and high PAI-1. Other abnormalities that were found among the RPL women were elevated levels of PAI-2 (like that made by the placenta) in nine and decreased urokinase-like plasminogen activator (u-PA) in six. The bottomline is that with these imbalances in the fibrinolytic system, decreased PA activity and/or increased PAI activity, there would be an increased tendency for blood clots to form and not be broken down. These findings led the authors to conclude that “activators and inhibitors of the fibrinolytic system are frequently abnormal in primary habitual aborters” and that “impaired plasmin dependent proteolysis (fibrin clot breakdown) in women might favor recurrent abortion by promoting fibrin deposition in early placental circulation or by limiting trophoblast development.”

Subsequent studies have supported and extended the findings and conclusions above. In 1999, Glueck and colleagues (Metabolism 1999;48:1589-95) found a significant correlation in women with polycystic ovary syndrome (PCOS) between elevated levels of PAI-1, early pregnancy loss, and no live births. These authors concluded it “is a predominant independent significant positive reversible risk factor for miscarriage in women with PCOS.” In 2003, Dossenabch-Glaninger, et al. (Clin Chem 2003;49:1081-6) evaluated 49 women with a history of two consecutive, or 3 to 6 nonconsecutive, early pregnancy losses compared to 48 women without a history of pregnancy loss for several genetic variants of the coagulation system. They found that homozygosity for PAI-1 or the factor XIII 34 Leu polymorphisms or compound heterozygous status (both of these polymorphisms in the same individual) of these same mutations significantly increased the risk for early pregnancy loss (OR = 2.4; 95% CI, 1.1-5.5).

In our last post, we mentioned that PAI-1 produced by vascular endothelial cells is induced by angiotensin II which is generated by the action of angiotensin I converting enzyme (ACE) and that autoantibodies directed against the angiotensin II type 1 receptor (AT1) found in preeclamptic women was associated with associated with increased production of PAI-1 (Xia, et al., J Soc Gynecol Invest 2003;10:82-93). Along the same lines, Buchholz and colleagues (Hum Reprod 2003;18:2473-7) studied the ACE deletion(D)/insertion(I) and the PAI-1 4G/5G polymorphisms in women with RPL, both of which are associated with increased ACE and PAI-1 expression, respectively. Comparing 184 women with a history of two or more consecutive spontaneous abortions with 127 women who had term pregnancies and no early losses, they found that homozygosity for the D allele of the ACE gene (D/D) was significantly correlated with RPL and the presence of the PAI-1 4G/4G homozygous state further increased PAI-1 levels and risk for early pregnancy loss. As a consequence of these findings, the authors recommended “the incorporation of these two polymorphisms into the spectrum of thrombophilic mutations which should be analyzed in individuals with recurrent spontaneous miscarriages.”

In more recent studies, Glueck and colleagues (Metabolism 2005;54:1345-9) reported that even among women who had had live births, if they had also had a spontaneous abortion, they were at greater risk than women who had never lost a pregnancy for having elevated levels of PAI-1 (33% vs 18%) and for the presence of other aberrations of the coagulation cascade: presence of factor V Leiden homozygosity (15.2% vs 1.6%) and elevated levels of factor VIII (31% vs 18%). These findings carried over to similar observations in women with PCOS (Glueck, et al., Metabolism 2006;55:345-52). In this study they assessed the association of PAI-1 levels in 430 women with PCOS who were divided into the following groups: 1) women who had live births only (n = 208); 2) women who had one or more live births and one or more first trimester losses (n = 111); 3) women who had only had first trimester miscarriages (n = 71). They found that “PAI-1 activity was positively associated with first-trimester miscarriage (p = 0.004)” … and “for each 5 IU/mL increment in PAI-1 activity, the risk being in an adverse first-trimester miscarriage …increased (OR, 1.12; 95% CI, 1.04-1.20).” In the same study, they also evaluated the association of the PAI-1 4G polymorphism in 921 women with PCOS compared to 126 normal females and again demonstrated (although the difference was not as dramatic in these women with a more heterogeneous obstetrical history – 78% in the PCOS group compared to 69% in controls) the 4G allele “is more common in women with PCOS than in normal women and, in concert with obesity, hyperinsulinemia, and hypertriglyceridemia, contributes to treatable, hypofibrinolytic, miscarriage-promoting, high PAI-1 activity.”

Coulam and colleagues (Reprod Biomed Online 2006;12:322-7) compared the prevalence of ten thrombophilic gene mutations in 42 women with a history of recurrent implantation failure after IVF embryo transfer with 20 fertile women. They found that the women with implantation failure had a significantly higher prevalence of PAI-1 4G/5G polymorphisms than controls (P = 0.007). Although they found no significant differences in the prevalence of any other single gene mutation, they did find “the prevalence of total gene mutations among patients with implantation failure was significantly higher than among controls. More than 3 gene mutations among the 10 genes studied were observed in 74% of women with implantation failure” compared to 20% of controls (P = 0.0004). They “concluded that inherited thrombophilias are associated with implantation failure” and this highly significant “association is manifest by totatl number of mutations as well as with PAI-1 mutations.”

Using the same approach, Coulam’s group also reported (Am J Reprod Immunol 2006;55:360-5) a comparison between 150 women with two or more recurrent pregnancy losses and 20 fertile women with no history of pregnancy losses. In this study they also found that there were “no differences in the frequency of specific gene mutations…however, the prevalence of homozygous mutations (59% vs 10%) and total gene mutations among patients with recurrent miscarriage was significantly higher than among controls.” As in their previous report, more than 3 mutations among the 10 genes studied were observed in a significantly higher