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Case Study:
Recurrent Spontaneous Abortion

ID: 31 year old healthy female with recurrent spontaneous abortion.

HPI: Patient is G5 P1 TAB1 SAB3. Her first pregnancy (1993) was electively terminated. She was pregnant again in 1995 and delivered a healthy baby at thirty-three weeks gestation. The next three pregnancies (1/99, 4/99 and 10/99) were spontaneously aborted at approximately six weeks gestational age. The fourth pregnancy (4/99) had sixty-four chromosomes. Obstetrical work-up included hysterosalpingogram, laparoscopy, endometrial biopsy, cervical cultures, and parental chromosomes. All of the studies were normal.

Patient has complained of bilateral knee pain, pleuritic chest pain, photosensitivity and occasional rash on the chest since her late teens. There has been no alopecia, malar rash, mucocutaneous ulceration, dry eyes or mouth, Raynaud's, unusual headaches, motor or sensory deficits, shortness of breath, gastrointestinal complaints or any other symptoms.

PMH: No known autoimmune disease

PSH: Negative except for reproductive system evaluation

FH: Father had a MI at age 39. He had no other risk factors for heart disease. No member had cerebral vascular accident, pulmonary embolus, deep venous thrombosis, diabetes mellitus type I, thyroiditis, systemic lupus erythematosus (SLE), recurrent miscarriage or any other autoimmune disease.

Laboratory Evaluation

  • Maternal antipaternal leukocyte antibodies: T-cell 1.1% and B-cell 15.0%
  • Antiphospholipid antibody (APA) panel (CL, PE, PI, PA, PG, PS): cardiolipin (IGM) positive
  • Antinuclear antibody (ANA) panel (ANA titer, dsDNA, Sm, Rnp, Ro, La): negative
  • Thyroid antibodies (antithyroglobulin, antimicrosomal): normal
  • Lupus anticoagulant: elevated (43; normal <40)
  • Immunophenotype:
    • NK (CD16+/CD56+) lymphocytes: normal (12)
    • Cytotoxic (CD5+) B-cells: normal (7)
  • Natural killer cells with IVIg: normal NK cell activity, stimulates with mitogen
  • HLA DQ alpha genotype, wife: 3, 4.1
  • HLA DQ alpha genotype, husband: 4.1, 4.1

Impression - Recurrent Spontaneous Abortion (RSA)

  1. Knee pain, pleuritic chest pain, photosensitivity, rash on chest
  2. Low blocking antibodies
  3. APA positivity
  4. Lupus anticoagulant prolonged
  5. DQ alpha sharing

Treatment Course
Patient received three paternal white cell immunizations (PLI), each PLI four weeks apart, before blocking antibody levels were optimized (55.1 and 63.9%). Two weeks prior to the cycle of planned conception, patient was instructed to take aspirin 81 mg QD and heparin 5000 units sq BID for APA positivity and lupus anticoagulant prolongation. Both medications were continued through week thirty-four gestation. Prednisone 10 mg BID was started concomitantly and tapered at twenty-four weeks gestation. Most immune studies were obtained every trimester to determine whether treatment required adjustment. The patient delivered a healthy boy at 38 weeks gestation.

Discussion
Over 50% of women who have recurrent spontaneous abortions have no anatomic, chromosomal, hormonal or infectious etiology for these occurrences. Studies have shown that these women lack maternal anti-paternal leukocyte antibodies as measured by flow cytometry. These antibodies, which coat the paternal antigens found on the placenta, are necessary to prevent the mother from aborting the fetus in much the same way that a patient would reject any type of graft. In addition, the antibodies stimulate the placenta to grow via release of cytokines. Kiprov showed (1992) that the vast majority of multiparous women have these blocking antibodies; whereas, age matched patients with recurrent miscarriages do not. Recent data from Matsubayashi, et. al. has shown that blocking antibodies measured by flow cytometry is an excellent predictor of pregnancy success (2000). Treatment using PLI has been used in humans since 1979 for this problem. Mowbray was the first to demonstrate in a randomized double-blinded study that PLI is an effective treatment (1985). After controversy arose following the publication of two poorly designed studies in 1991, the American Society of Reproductive Immunology conducted two independent meta-analyses of the world's data, and published the results in December 1994. Both analyses demonstrated the statistically significant benefit of PLI. Notable was that the only criteria for treatment was the clinical manifestation of recurrent miscarriage; only superficial autoimmune screening was done for exclusionary criteria. Another analysis, also published December 1994, showed that efficacy of PLI is enhanced if the patients who are immunized lack blocking antibodies before the treatment, and develop them after therapy.

Phospholipid molecules are normal components of all cell membranes. Antibodies to phospholipids (APA) have been implicated in numerous disease states (CVA, MI, PE, DVT, RSA), generating much academic interest. APAs 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 (IUGR).

Some phospholipid molecules—particularly phosphatidylserine and phosphatidylethanolamine—have adhesive properties. They allow cells to fuse so that in the placenta, cytotrophoblasts become syncytiotrophoblasts, which regulate nutrients to the fetus. A study on the formation of syncytia in the trophoblastic cell line Be-Wo discovered that monoclonal antibodies to phosphatidylserine (but not to cardiolipin) inhibited the formation of syncytia in-vitro. Furthermore, placentas from patients with APA who miscarried had a high percentage of immunoglobulin M (IgM) APA bound to the syncytia. Interference with cell adhesion by APA may predate clotting abnormalities.

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 has 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 (81mg qd) and prophylactic, minidose heparin. Heparin 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 has been shown in studies not to affect the fetus. Aspirin inhibits cyclooxygenase and the formation of thromboxanes, allowing prostacyclin to act unopposed, which results in vasodilation. Treatment is more effective when medication, if indicated, is started prior to conception and continued throughout pregnancy.

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. 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 are 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, a large protein molecule. In addition, the placenta contains Beta2-dehydrogenase, which metabolizes this steroid, inactivating it. 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. The body is dynamic, and antibody levels may change over time. Patients who develop new autoantibodies during pregnancy have a more guarded prognosis.

The use of IVIg has become increasingly more important as scientists discover the uses of this biopharmaceutical in cases of recurrent pregnancy loss. IVIg has been shown to block the immunoglobulin Fc receptor on monocytes, alter T-cell subsets, modulate cellular immune responses, reduce NK cytotoxicity, and cause humoral immunity (ANA and APA) suppression. Normally, NK cells are down regulated in the early stages of a pregnancy. However, significantly higher levels of CD56+CD16+ NK cells (level=17%) were demonstrated in women who miscarried vs. those who delivered (level=12%) the index pregnancy. The success of IVIg has been shown; women with elevated NK cells, but who were treated with IVIg, were able to deliver successfully in 86.3% of the cases.

HLA DQ alpha matching may influence polyclonal B-cell activation, and up regulation of NK cells. Nelson was the first to notice that HLA DQ alpha incompatibility between mother and fetus was highly correlated with remission of rheumatoid arthritis during pregnancy. Subsequently, Ober, et. al. showed that women who miscarry despite "optimal" allo and autoimmune therapy are DQ alpha identical with the fetus. Kwak, et. al. demonstrated an exacerbation of antiphospholipid antibodies in patients with HLA DQ alpha compatibility before they miscarry. The conclusion is that HLA DQ alpha compatible mother and fetus may increase the synthesis of TH-1 cytokines and NK cells in the trophoblast, exacerbating autoimmune phenomena that can lead to a miscarriage. The clinician must consider empiric use of medications in early pregnancy to combat this occurrence.

References

Beer AE, Kwak JYH., "What is the evidence for immunologic pregnancy loss?" Lymphocyte immunization the supportive view In: Mowbray JF, Chaoaut G, eds. Molecular and Cellular Biology of Maternal-Fetal Relationship. Paris: Inserm Libbey.

Chong, P., Matzner, W., et. al., "Characterization of Antiphospholipid Antibodies in Women with Recurrent Fetal Miscarriage," Journal of Reproductive Medicine, 1994. Jan; 39(1): 27-30.
• Download article (PDF 193K)

Chong, PJ, Matzner, WL, Ching, WT., "Immunology of Recurrent Spontaneous Abortion: A Review," The Female Patient. 1995. Feb; 20(2): 46-53.
• Download article (PDF 59K)

Collins, John, Roberts, Robin, Scott, James., "Reports of Independent Analyses of Data From the Worldwide Prospective Collaborative Study on Immunotherapy for Unexplained Recurrent Spontaneous Abortion," Amer J. Reprod. Immunol. 1994; 32: 275-280.

Coulam CB. Coulam CH., "Update on immunotherapy for re-current pregnancy loss," Am J Reprod Immunol.1992;27: 124-127.

Coulam, CB, Goodman, C, et. al., "Systemic CD56+ cells can predict pregnancy outcome," Amer J. Reprod. Immunol. 1995; 34: 93-99.

Daya, Salim, Gunby, J. and The Recurrent Miscarriage Immunotherapy Trialists Group., "The Effectiveness of Allogeneic Leukocyte Immunization in Unexplained Primary Recurrent Spontaneous Abortion," Amer J. Reprod. Immunol. 1994; 32: 294-302.

Doherty R, Slubblefield P. Dostal-Johnson D, Day G., "While blood cell immunization and anticardiolipin antibody levels in women with recurrent miscarriages," Fertil Steril. 1992;58: 199-207.

Harris EN, Gharavi AE, Hughes GRV., "Antiphospholipid antibodies Clin Rheumatol," Dis. 1985; 11:591 -609.

Holzgreve W, Garritsen HS. Ganshirt-Ahlert D., "Fetal cells in the maternal circulation", J Reprod Med. 1992;37 (41): 418.

Kiprov, et. al., J. Immunol Immunopharmacol. 1992; 12: 108.

Kovats S, Main EK, Librach C, et al., "A class I antigen, HLA-G, expressed in human trophoblasts," Science. 1990;248: 220 223.

Koyama M, Saji F, Takahashi S. et al., "Probabilistic assessment of the HLA sharing of recurrent spontaneous abortion couples in the Japanese population," Tissue Antigens. 1991;37:211-217.

Kwak, JYH, Kwak, FMY, et. al., "Elevated Peripheral Blood Natural Killer Cells Are Effectively Downregulated by Immunoglobulin G Infusion in Women With Recurrent Spontaneous Abortions," Amer J. Reprod. Immunol. 1996; 35: 363-369.

Kwak, JYH, Gilman-Sachs, A, et. al., "Reproductive outcome in women with recurrent spontaneous abortions of alloimmune and autoimmune etiologies: pre vs. post conception treatment," Am J Obstet Gynecol. 1992; 166: 1787-1798.

Kwak, JYH, Quilty, EA, et. al., "Intravenous immunoglobulin G infusion therapy in women with recurrent spontaneous abortions of immune etiologies," J Reprod Immunol. 1995; 28: 175-188.

Lyden BS, Ng AK, Rote NS., "Modulation of phosphatidylserine epitope expression on BeWo cells during forskolin treatment," Amer J. Reprod. Immunol. 1992;27:24.

Matsubayashi, H, Maruyama, T, Ozawa, N, et.al., "Anti-Paternal Antibodies by Flow Cytometry in the Management of Alloimmunization on Recurrent Miscarriages" Amer J. Reprod. Immunol. 2000; 44: 284-288.

Matzner B, White I., "Solving reproductive problems with a computer," Physicians and Computers. 1991;9:283.
• Download article (PDF 234K)

Matzner, W., Chong, P., et. al., "HLA Antigen Profiles in Women with Recurrent Fetal Miscarriage," Journal of Immunology, 1993. Apr; 150(8): 200A.

Mowbray, JF, Gibbing, C, Liddell, H, Reginald, PW, Underwood, JL, Beard RW., "Controlled trial of treatment of recurrent spontaneous abortion by immunization with paternal cells," Lancet. 1985; I: 941-943.

Nelson, JL, Hughes, KA, Smith, AG, et. al., "Maternal-fetal disparity in HLA class II alloantigens and the pregnancy-induced amelioration of rheumatoid arthritis," N Engl J Med. 1993; 329: 466-471.

Ober, C, Steck, T, van der VenK, et. al., "MHC Class II compatibility in aborted fetuses and term infants of couples with recurrent spontaneous abortion," J Reprod Immunol. 1993; 25: 195-207.

Reed E, Beer AE, Hutcherson H. et al., "The alloantibody response of pregnant women and its suppression by soluble HLA antigens and anti-idiotypic antibodies," J Reprod Immunol. 1991 ;20: 115-128.

Reznikoff-Etievant MF. Bonneau JC, Alcalay P, et al., "HLA antigen sharing in couples with repeated spontaneous abortions and the birthweight of babies in successful pregnancies," Amer J. Reprod. Immunol. 1991 ;2.5 :25-27.