Dr. William Matzner Blog

Excerpt: Characterization of Antiphospholipid Antibodies in Women with Recurrent Spontaneous Abortions

Dr. William Matzner, Simi Valley, California

Characterization of Antiphospholipid Antibodies in Women with Recurrent Spontaneous Abortions

This is an excerpt of an article originally published in The Journal of Reproductive Medicine and was co-authored by Dr. William Matzner. The full article is available here.

Introduction

It is estimated that approximately 40% of women with systemic lupus eythematosus (SLE) will have antibodies to negatively charged phospholipid, with reported ranges of 30% to 70% having suffered thrombotic events. In 1986 a group of women without known autoimmune diseases who had recurrent spontaneous abortions (RSA) or vascular thrombosis was described. These women had antibodies to a negatively charged phospholipid, cardiolipin, with high titers primarily of the IgG isotype. The mechanisms of action defined were platelet membrane damage, endothelial wall injury, inhibition of prostacyclin and inability to activate protein C; thus, RSA would occur via placental vascular insufficiency. In 1985 Harris showed that anticardiolipin antibody (aCL) bound equally to all negatively charged phospholipids. Subsequent studies that addressed issues of antiphospholipid antibodies (aPLs) therefore assumed nondifferential binding or concentrated only on aCL.

In 1985 Lockshin et al demonstrated that the level of aCL was useful as an early predictor of fetal distress or death in patients with SLE. Recent work has implicated antiphosphoserine antibody, predominantly of the IgM isotype, as an inhibitor of placental formation and a cause

of RSA.

In the study described below, aPLs were characterized in a group of women who have suffered recurrent fetal loss.

Materials and Methods

Patients

Three hundred fifty-two patients with a history of two or more consecutive spontaneous pregnancy losses were evaluated. They were premenopausal (aged 2145; mean, 39), and all were recruited between March 1991 and May 1992. Women with collagen vascular disease were excluded. Phospholipid antibodies of the IgM and IgG isotypes to cardiolipin, phosphoethanolamine, phosphoinositol, phosphoserine, phosphatidic acid and phosphoglycerol were measured.

Controls

The control group consisted of 43 people without known immunologic or rheumatologic diseases. None of the women in the control group had suffered RSA or any other thromboembolic phenomena.

aPL Assay

The assay used has been described previously. Briefly, six purified phospholipids were coated separately on Immulon 2 96-well enzyme-linked immunosorbent assay plates overnight. The plates were blocked the next day with phosphate-buffered saline (PBS) and 10% newborn calf serum for two hours and then washed (Biotech BT500) in PBS.

Fifty microliters of patient serum was added to the appropriate wells and incubated for one hour followed by a second wash with PBS. Alkaline phosphatase-conjugated goat antihuman IgG and IgM was incubated for an hour, then washed in PBS. Sigma Substrate 104 in diethanolamine buffer was added to the wells and incubated for 30 minutes at 37°C, and the reaction was stopped with NaOH. The trays were read in a BioTech BT2000 microtiter reader at 405 nm. Delta optical densities were calculated by subtracting out the background (wells without the phospholipid antigens).

The results were compared to the mean of the delta optical densities for the control group. Positivity was defined as 3 SD above the mean of the controls.

Results

The prevalence of one or more antibodies to any of the six phospholipids was 59.1% (208/352) in the study population. In the control group, only 4.6% (2/43) had a positive aPL.

Two hundred eight patients had 439 antibodies of the IgG or IgM isotype to the phospholipids. The most frequently identified aPLs in order of decreasing frequency were to phosphoserine, 20.5% (90/439); phosphoethanolamine, 19.1% (84/439); phosphatidic acid, 18.7% (82/439); cardiolipin, 16.4% (72/439); phosphoglycerol, 16.2% (71/439); and phosphoinositol, 9.1% (40/439). In the two positive controls there were 2 anitibodies, both of the IgG isotype, 1 each to cardiolipin and phosphatidic acid. In the RSA patients, 75.2% (330/439) of the antibodies were of the IgM isotype, and 24.8% (109/439) were of the IgG isotype.

Of all of the patients studied, 18.2% (64/352) had aCL versus 40.9% (144/352) with any other combination excluding aCL. Of all the patients with any aPL, 30.8% (64/ 208) were to cardiolipin. Eighty-one patients had antibodies to only one epitope: 37.0% (30/81) were to phosphoethanolamine, 14.8% (12/81) to phosphatidic acid and 16.0% (13/81) to cardiolipin. Of this group, 83.9% (68) were IgM and 16.1% (13) were IgG isotypes.

The remainder of the patients (132) had antibodies to multiple epitopes. They had 358 antibodies of the IgG or IgM isotype. In this group, in order of decreasing frequency, were antibodies to phosphoserine, 22.9% (82/ 358); phosphatidic acid, 19.6% (70/358); phosphoglycerol, 18.2% (65/358); cardiolipin, 16.5% (59/358); phosphoethanolamine, 15.1% (54/ 358); and phosphoinositol, 7.8% (28/358). In these patients, 73.2% (262/358) of the antibodies were of the IgM and 26.8% (96/358) of the IgG isotype.

About William L. Matzner, M.D., PhD, FACP

Dr. William Matzner works in the area of healthcare economics consulting at Healthcare Analytics, LLC, in California. He graduated Phi Beta Kappa from Stanford University. He received his M.D. with Honors from Baylor College of Medicine. In 1988, he was the Solomon Scholar for Resident Research at Cedar Sinai Medical Center. Dr. Matzner subsequently was awarded a PhD in Neuro Economics from Claremont Graduate University. He is board certified in Internal Medicine and Palliative Medicine. He has researched and published extensively on the issue of reproduction and immunology in medical literature. He has been in private practice since 1989, specializing in Reproductive Immunology and Internal medicine.

Website: https://drwilliammatzner.com

Consulting Website: https://healthcareanalytics.biz

LinkedIn: https://www.linkedin.com/in/william-matzner-md-phd-mba-60219730

Blog: https://drwilliammatzner.blogspot.com

News: https://hype.news/dr-william-matzner/

News: https://hippocratesguild.com/dr-william-matzner

News: https://medicogazette.com/dr-william-matzner

William Matzner, MD (Simi Valley, California), has been practicing medicine since 1989, Internal Medicine and Reproductive Immunology. M.D. with Honors from Baylor College of Medicine.

Excerpt: Immunology of Recurrent Spontaneous Abortion

William Matzner, MD, Simi Valley, California

This is an excerpt of an article originally published in The Female Patient and was co-authored by Dr. William Matzner. The full article is available here.

Immunology of Recurrent Spontaneous Abortion

Introduction

The immunology of reproduction is a dynamic field, with data forthcoming exponentially. Immune mechanisms are operative in infertility, endometriosis, eclampsia/preeclampsia, miscarriage, and other aspects of reproduction. This paper focuses on the immunology of recurrent spontaneous abortion (RSA). Classically, a patient is considered to have RSA if she has had three or more consecutive miscarriages, but many clinicians are now evaluating couples after two consecutive losses.

The causes of RSA have been classified as infection (1%), anatomic abnormalities (5% to 10%), lutealphase defect (5% to 20%), chromosomal abnormalities (7% to 50%), immune mechanisms (50%), and unknown (15%). Some women have multiple reasons for RSA. A workup comprising ultrasonography, hysterosalpingography, laparoscopy, endometrial biopsy, parental and fetal chromosome analysis, cervical culture, and progesterone testing would explain only about 50% of the pregnancy losses. There is strong evidence that the remainder of miscarriages are mediated by immune mechanisms.

The uterus is an enigma. Despite a full complement of immunocompetent cells, it allows the fetal allograft to thrive for 40 weeks. During pregnancy, the fetoplacental unit orchestrates immune mechanisms via T and B Iymphocytes, natural killer cells (NK), a variety of soluble immunoregulatory factors (cytokines), and antibodies. To a significant degree, the interaction between maternal and fetoplacental tissue and the immune system will determine whether a pregnancy succeeds. Three antibodies are critically important to pregnancy maintenance: maternal antipaternal leukocyte antibodies (APLA) (ie, blocking antibodies), antiphospholipid antibodies (APA), and antinuclear antibodies (ANA).

When the immune system is the cause of miscarriage, the mother has a 30% chance of having a successful pregnancy without intervention after 3 miscarriages, a 25% chance after 4 miscarriages, and a 5% chance after 5 miscarriages. With proper treatment, the overall success rate has been reported at 70% to 85% in parity and agematched controls.

Antipaternal Leukocyte Antibodies

APLA are antibodies that mask paternal human leukocyte antigens (HLA) found on the fetus from maternal immune effector cells. Genes that code for HLA or tissue type are located on chromosome 6. HLA consist of class I and class II antigens. Class I antigens, which include the A, B, and C loci, are found on all nucleated cells and platelets and are the only HLA expressed on nonactivated T Iymphocytes. More recently, another class I HLA, designated G, has been identified on cytotrophoblasts versus syncytiotrophoblasts, which do not express any HLA. However, studies using the polymerase chain reaction have shown that the placental barrier is not impervious to tissue, so that maternal cells have been found in the fetal circulation and fetal cells in maternal circulation.

In the RSA couple, APLA levels should be ascertained prior to conception using cell-flow cytometry. The husband’s Iymphocytes are combined with the wife’s serum (which would contain APLA if present), and incubated with fluorescent markers. The entire mixture is placed into the cytometer, which utilizes laminar flow fluidics and argon lasers. Under laser illumination, cells that have APLA attached will fluoresce. The emission will be captured by photomultiplier tubes and transferred to a computer that digitizes the signal.

Treatment involves immunizing the mother with concentrates of paternal Iymphocytes so that the signal is amplified approximately 10,000 times the level normally seen in early pregnancy. Paternal leukocyte immunization (PLI) treatments are usually administered 4 weeks apart. Four weeks after the second immunization, the APLA level is remeasured.

Antiphospholipid Antibodies

Phospholipid molecules are normal components of all cell membranes. Antibodies to phospholipids have been implicated in numerous disease states, generating much academic interest. APA are capable of vascular compromise via damage to vascular endothelium and platelet membrane by inhibiting prostacyclin (vasodilator) and interfering with the activation of protein C. The result is increased platelet adhesion and a relative rise in thromboxane (vasoconstrictor), resulting in a milieu conducive to thrombotic events. In the uteroplacental circulation these insults translate into fetal demise or intrauterine growth retardation.

With each pregnancy loss, there is a 10% chance that the mother will develop an antibody to a phospholipid molecule, and the effect is cumulative. Most women with APA are asymptomatic, but some have underlying autoimmune tendencies and should be evaluated appropriately. Although there is a high incidence of APA in patients with systemic lupus erythematosus (SLE), there is a significant population who have APA but no other disease. The diagnosis assigned to patients with thrombotic events in the presence of APA is primary antiphospholipid antibody syndrome.

Treatment of APA involves the use of low-dose (baby) aspirin and prophylactic heparin, which is a large molecule that cannot cross the placenta. Heparin activates the formation of antithrombin III, which interferes with the coagulation cascade. Although aspirin can traverse the placenta, the dose is small and usually does not affect the fetus. Aspirin inhibits cyclooxygenase and the formation of thromboxanes, allowing prostacyclin to act unopposed. Treatment is more effective when medication, if indicated, is started prior to conception and continued throughout pregnancy.

Antinuclear Antibodies

There is an increased prevalence of RSA patients who demonstrate ANA compared with parity- and age-matched nonaborters. What causes these antibodies to be synthesized is currently under investigation, but there appears to be a genetic susceptibility dictated by the HLA tissue type. This is compounded by the production of autoantibodies like ANA with fetal demise. The disease typically associated with ANA is SLE, which confers a much higher miscarriage rate than that of the general population— approaching 50% in patients with active disease. Although most women with RSA do not fulfill the American College of Rheumatology criteria for SLE, many exhibit lupus-like tendencies. Polyclonal B cell activation appears to be more common in these patients. Although the exact mechanisms whereby ANA contribute to miscarriage is unknown, placental pathology studies reveal inflammatory changes in the uterine and placental tissue (villitis) and vasculitis.

When ANA are present in the context of RSA, prednisone is recommended to suppress the inflammatory process and stabilize cell membranes. Prednisone does not cross the placenta easily because it is highly bound to albumin, which is a large protein molecule. In addition, the placenta contains Beta2-dehydrogenase, which metabolizes this steroid. Suppression of the fetal adrenal axis has not been reported. When indicated, prednisone is instituted prior to conception. With treatment, there is an 80% to 85% chance of successful term pregnancy. As the body is dynamic, antibody levels may change over time. Patients who develop new autoantibodies during pregnancy have a more guarded prognosis.

Conclusion

Failure of maternal response to the fetal allograft, as well as the production of autoantibodies, can result in repetitive pregnancy loss. Contrary to popular belief, miscarriage is not a benign process, as the patient may develop autoantibodies. Fortunately, these problems are easily identified and amenable to treatment. Miscarriage due to immune dysfunction is largely preventable today, and couples desiring parenthood should be given appropriate consideration and evaluation.