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Case Study: Multiple Failed IVF

ID: 38 year old healthy female with multiple failed IVF

HPI: Patient is G0 P0. She was evaluated in England, assigned the diagnosis of unexplained infertility (laparoscopy and endometrial biopsy in 1996 were normal) and subsequently underwent three IVF cycles (2/9, 5/97, 10/97) that did not result in pregnancy. Between 12/97 and 5/98 the patient had multiple stimulated (Clomid) IUI cycles without success.

Patient had a history of photosensitivity, tingling sensation in the hands and occasional dry eyes. There were no complaints of arthritis, myalgias, Raynaud's, malar rash, mucocutaneous ulceration, alopecia, motor or sensory deficits, unusual head aches, chest pain, shortness of breath, GI complaints or any other symptoms.

Patient hoped to undergo another IVF cycle but wanted to increase the probability of success. Immune work-up at RIA commenced 7/98 (see Laboratory Evaluation).

PMH: No known autoimmune disease.

PSH: Negative except for reproductive system evaluation.

FH: No early cerebral vascular accident, myocardial infarction, pulmonary embolus or deep venous thrombosis. There is no diabetes mellitus I, thyroiditis, systemic lupus erythematosus (SLE), recurrent miscarriage, or any other autoimmune disease.

Laboratory Evaluation

  • Maternal antipaternal leukocyte antibodies: T-cell 0.1% and B-cell 1.3%
  • Antiphospholipid antibody panel (APA) panel (CL, PE, PI, PA, PG, PS): phosphoinositol borderline
  • Antinuclear antibody (ANA) panel (ANA titer, dsDNA, Sm, Rnp, Ro, La): negative
  • Thyroid antibodies (antithyroglobulin, antimicrosomal): negative
  • Lupus anticoagulant: normal
  • Immunophenotype: NK (CD16+/CD56+) lymphocytes: normal (14)
  • Cytotoxic (CD5+) B-cells: elevated (9)
  • Natural killer cell activity with IVIg: normal NK cell activity, stimulates with mitogen
  • HLA DQ alpha genotype, wife: 3, 4
  • HLA DQ alpha genotype, husband: 3, 4

Impression - Multiple failed IVF

  1. Low blocking antibodies
  2. APA positivity
  3. Cytotoxic B-cells elevated
  4. DQ alpha sharing

Treatment course
Patient received two paternal white cell immunizations (PLI), four weeks apart, with no discernible change in her blocking antibody levels. She received two more PLI that moved the T-cell and B-cell levels to 28 and 53%, respectively. Two weeks prior to the transfer, patient was instructed to take aspirin 81 mg/d and heparin 5000 units sq BID for + APA. Due to the DQ alpha sharing and elevated cytotoxic B-cells, IVIg 500 mg/kg X 1 dose was infused ten days prior to the transfer. The patient delivered a healthy girl at 36 weeks gestation following premature rupture of membranes.

There appears to be a continuum in the clinical manifestations of patients with immune-mediated reproductive failure. The spectrum proceeds from failed IVFs and unexplained infertility to recurrent miscarriages, and then to intrauterine growth retardation (IUGR) and pre-eclampsia. In the above example, this patient may be having very early pregnancy losses, and not failed IVFs.

It has been shown that patients who have recurrent miscarriage lack maternal anti-paternal leukocyte (blocking) antibodies as demonstrated by flow cytometry. 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 Matusbayashi, 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. 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.

Eleven out of twelve studies have demonstrated a significant increased prevalence of APAs in patients undergoing failed IVFs. However, there have been inconsistencies as to whether standard miscarriage treatment with aspirin and heparin would be beneficial to these patients. Geva, et. al. found that serologic positivity was more common in 50 women (22%) who had >3 failed embryo transfers vs. 40 matched women who had conceived in <2 attempts (2.5%). Birkenfeld measured LA and anticardiolipin antibodies (aCL) in women undergoing embryo transfer (ET) for tubal infertility. Sixteen (29%) of 56 women who didn't conceive had greater than one autoantibody vs. 0/14 (0%) who successfully implanted.

A very large study from Pacific Fertility Center patients showed a very significant improvement when aspirin and heparin were administered. The authors of the study point out that the patients were limited to women undergoing IVF because of female organic pelvic pathology. Furthermore, the treatment was effective only when aspirin and heparin was administered full two weeks prior to the transfer. APA positivity was based upon the assay used at RIA laboratory that measures three isotypes of three phospholipid epitopes. Studies that have failed to demonstrate an efficacy either used an inadequate assay (Denis, et. al.), used the wrong patient subpopulation for its proper administration (Denis, et. al.), only measured aCL or LA instead of the complete APA panel, or administered the heparin/aspirin with an inadequate dose or time before IVF transfer.

Intravenous immune globulin (IVIg) has been used in several studies on patients undergoing IVF. De Placido et. al. found a three fold increase in embryo implantation rates (17.7% vs. 8%) after using IVIg. Kleinstein et. al. demonstrated a six fold enhancement in clinical pregnancy rates (50% vs. 8%) when IVIg was given. Coulam, et. al. found if a woman previously fertilized more than 50% of the oocytes retrieved and produced greater than three embryos, IVIg helped 56% become pregnant. Our laboratory conducted four further studies to elucidate specific indications for administration of IVIg in IVF patients. The first, a randomized study, found that patients with antithyroid antibodies had a 51% success rate when IVIg was administered, as opposed to 27% for controls. The second study focused on patients with four or more failed IVFs. In this group, IVIg lead to a 42% birth rate if these patients were APA seropositive, but only 19% birth rate if seronegative. The third study followed APA seropositive patients serially in successive IVF cycles. It demonstrated that IVIg was needed in addition to aspirin and heparin when patients APAs were IgG or IgM isotypes of phosphatidylserine (PS) or phosphatidylethanolamine (PE). The last study in this series showed a very high correlation between IgM and IgG PS and PE positivity, and elevation of natural killer (NK) cell activity as demonstrated by the K562 killing assay. One can therefore conclude that the IVIg given in the PE/PS+ study was likely affecting NK cell activity, accounting for the high success rate. A recently published randomized studied that failed to show efficacy of IVIg therapy in IVF patients used an inadequate dose given too late in the cycle (the day of transfer). The authors (Stephenson, et. al.) admitted in their discussion that this could be the reason why their study failed to show a difference.

Since the first study by Komlos, there have been 18 studies demonstrating increased HLA sharing in patients with recurrent pregnancy loss. Ho, et. al., published a study showing that this same phenomena occurs in failed IVF patients. Seventy-six couples with unexplained infertility underwent IVF. Thirty-four had successful pregnancies, 36 did not become pregnant, and 6 had SABs. The couples who failed treatment shared three of the A, B, DR or DQ antigens, or two of the B, DR and DQ antigens (p=.015).

The body of research has lead the American Society of Reproductive Immunology to officially describe the Reproductive Autoimmune Failure Syndrome (RAFS) for these patients. The diagnosis of RAFS is considered when a woman has at least one clinical criterion and two or more abnormal laboratory tests showing evidence of polyclonal B-cell activation. Clinical criteria include pregnancy wastage (embryonic loss, fetal loss, fetal growth impairment, severe pre-eclampsia, HELLP, herpes gestationis, abruptio placenta, or chorea gravidarum), unexplained infertility, and endometriosis. Laboratory criteria for RAFS include lupus anticoagulant, gammopathy, antiphospholipid antibodies (IgM, IgG, IgA), antinuclear antibodies, antihistone antibodies, and organ specific antibodies (antithyroid antibodies, antismooth muscle antibodies).


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Chong, PJ, Matzner, WL, Ching, WT., "Immunology of Recurrent Spontaneous Abortion: A Review," The Female Patient. 1995. Feb; 20(2): 46-53.
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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, Branch, DW, Clark, CA, Gleicher, N, Kitteh, W, Lockshin, M, Rote, N., "American Society for Reproductive Immunology: Report of the Committee for Establishing Criteria for Diagnosis of Reproductive Autoimmune Syndrome," Amer J. Reprod. Immunol. 1999; 41: 121-132.

Coulam, C.B., Krysa, L.W., and Bustillo, M., "(1994) Intravenous immunoglobulin for in-vitro fertilization failure," Hum. Reprod. 9, 2265-2269.

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.

Denis, AL, Guido, M, Adler, RR. et. al., "(1997) Antiphospholipid antibodies and pregnancy rates and outcome in in-vitro fertilization patients," Fertil. Steril., 67, 1084-1090.

De Placido, G., Zullo, F., Mallo, A., Capiello, F., Nazrro, A., Coacurci, N., Palumbo, G., "(1994) Intravenous immunoglobulin (IVIg) in the prevention of implantation failures," Ann NY Acad Sci, 734, 1-3.

Geva, E. Amt, A, Lerner-Gega, L, Yaron, Y, Daniel, Y., et. al., "Prednisone and Aspirin Improve Pregnancy Rate in Patients with Reproductive Failure and Autoimmune Antibodies: A Prospective Study," Amer. J. Reprod. Immunol. 1994; 43: 36-40.

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Kleinstein, J., Kanaga, O., Gips, H., and Kunzel, W., "(1994) Intravenous immunoglobulin increase clinical pregnancy rates in an IVF program," Soc.Gynec Invest; 41st annual meeting, abstr #P108.

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

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

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Sher, G, Feinman, M, Zouves, C, Kuttner, G, Maassarani, G, Salem, R, Matzner, W, Ching, W, Chong, P., "High Fecundity Rates Following In-vitro Fertilization and Embryo Transfer in Antiphospholipid Antibody Seropositive Women Treated with Heparin and Aspirin," Human Reproduction. 1994. Dec; 9(12): 2278-2283.
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Sher, G, Fisch, JD, Maassarani, G, Matzner, W, Ching, W, Chong, P., "Antibodies to phosphatidylethanolamine and phosphatidylserine are associated with increased natural killer cell activity in non-male factor infertility patients," Human Reproduction. 2000. 15(9): 1932-1936.
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Sher, G, Maassarani, G, Zouves, C, Feinman, M, Sohn, S, Matzner, W, Chong, P., Ching, W., "The Use of Combined Heparin/Aspirin and Immunoglobulin G Therapy in the Treatment of In Vitro Fertilization Patients with Antithyroid Antibodies," American Journal of Reproductive Immunology. 1998. 39(4): 223-225.
Download article (PDF 43K)

Sher, G, Matzner, W, Feinman, M, Maassarani, G, Zouves, C, Chong, P, Ching, W., "The Selective Use of Heparin/Aspirin Therapy, Alone or in Combination with Intravenous Immunoglobulin G in the Management of Antiphospholipid Antibody-Positive Women Undergoing In Vitro Fertilization," American Journal of Reproductive Immunology. 1998. 40(1): 21-30.
Download article (PDF 69K)

Sher, G, Zouves, C, Feinman, M, Maassarani, G, Matzner, W, Chong, P, Ching, W., "A Rational Basis for the Use of Combined Heparin/Aspirin and IVIG Immunotherapy in the Treatment of Recurrent IVF Failure Associated with Antiphospholipid Antibodies," American Journal of Reproductive Immunology. 1998. 39(6): 391-394.
Download article (PDF 46K)

Stephenson, M, Fluker, M., "Treatment of repeated unexplained in-vitro fertilization failure with intravenous immunoglobulin: a randomized placebo controlled Canadian trial," Fertil. Steril. 2000; 74 (6): 1108-1113.