Excerpt: A Rational Basis for the Use of Combined Heparin/Aspirin and IVIg Immunotherapy in the Treatment of Recurrent IVF Failure Associated with Antiphospholipid Antibodies

William Matzner, MD
William Matzner, MD, Simi Valley, CA
Excerpt: A Rational Basis for the Use of Combined Heparin/Aspirin and IVIg Immunotherapy in the Treatment of Recurrent IVF Failure Associated with Antiphospholipid Antibodies

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

INTRODUCTION

It has been demonstrated that antiphospholipid antibodies (APA) play a role in reproductive failure including recurrent pregnancy loss (1,2,3), unexplained infertility (1), pregnancy related hypertension (4,5) and intrauterine growth retardation (4).  Other studies (5,6,7) link APA to In Vitro Fertilization (IVF) or Embryo Transfer (ET) failure.  We previously reported on a negative correlation between APA positivity and IVF outcome and established a therapeutic relevance for the selective administration of mini-dose Heparin/Aspirin therapy (H/A) (8). In a subsequent study we demonstrated that IVIg was beneficial in a subset of women with a specific APA profile undergoing IVF (9).  Coulam et. al, reported that the use of IVIg prior to IVF resulted in a 56% success rate among a limited number of patients with multiple failed IVFs (12).  The purpose of this study was to help identify criteria for the administration of IVIg in patients who suffered repeated IVF failure.  Due to recent controversy regarding the use of H/A in patients undergoing IVF (15), we elected to treat all patients with these drugs eliminating the potential that this variable could impact outcome results when studying the effects of IVIg on these patients.

MATERIALS AND METHODS

PATIENT POPULATION

Eighty nine (89) consecutive women who fulfilled the study criteria were included in this study. The inclusion criteria were a) age <36 years, b) four or more failed IVF/ET, c) no male infertility, d) no ovum donation, e) no gestational surrogacy and, f) serum FSH concentration of <15 mIU/ml and a plasma E2 of <70 pg/ml on cycle day three.  All patients received gonadotrophin releasing hormone agonist (Lupron, Tapp Pharmaceuticals) for luteal phase pituitary down regulation, followed by menotropin therapy as previously described (10).  Starting on day two of controlled ovarian hyperstimulation, each patient received aspirin 81 mg po qd, and heparin 5000 U sq bid.  In addition, each patient received 20 gm of intravenous immunoglobulin (IVIg- Gammimune, Bayer Biological or Venoglobulin, Alpha Therapeutic Corp) 3-10 days prior to embryo transfer.

LABORATORY EVALUATION

All women underwent serum follicle stimulating hormone (FSH) and estradiol (E2) measurements (by radioimmunoassay) on day two or three of a prior menstrual cycle. All women underwent APA testing using an enzyme linked immunosorbent (ELISA) assay for IgM, IgG and IgA isotypes to six phospholipid epitopes (cardiolipin-CL, phosphoserine-PS, phosphoglycerol-PG, phosphoethanolamine-PE, phosphatidic acid-PA, and phosphoinositol-PI) as described previously in detail (11). Borderline positives were defined as >2 SD above the mean of normal controls, and positive values were defined as >3 SD above the mean of normal controls.  The control group for the APA assay consisted of non-infertility patients who had no history of clinical or subclinical autoimmune disease, or recurrent pregnancy loss.

Each time the ELISA assay was performed, both known negative and positive controls were run simultaneously for each isotype of every epitope.  (This was important to assess the performance of the antigen coated on each plate, the antibody conjugates, the pipetting technique, washing method, incubation times, incubation temperature and substrate).

Cervical or semen specimens were cultured for Ureaplasma, Chlamydia and Gonococcus, in all cases. Male partners all underwent semen analysis and both women and men had sperm antibody serologies measured using the indirect immunobead test.

DETERMINANTS OF OUTCOME

The number of babies born per transferred embryo was determined in order to  provide a measure of the viable implantation rate.  Multiple births and miscarriages were documented.  A successful IVF outcome was defined as a live birth.

STATISTICAL METHODS

Data was placed into two-by-two tables:  An analysis between and within groups were performed using the Chi Square Test for significance. P values below 0.05 were considered to indicate statistical significance.  Analysis was performed using the CHITEST and CHIINV functions for Microsoft Excel 97 for Windows 95. 

Rest of the article is available at the link provided above.

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. 

Consulting Website: https://healthcareanalytics.biz 


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: The Use of Combined Heparin/Aspirin and Immunoglobulin G. Therapy in The Treatment of IVF Patients with Antithyroid Antibodies


William Matzner, MD
Dr. William Matzner, Simi Valley, CA
Excerpt: The Use of Combined Heparin/Aspirin and Immunoglobulin G. Therapy in The Treatment of IVF Patients with Antithyroid Antibodies

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

INTRODUCTION

A relationship between antithyroid antibodies (ATA) and reproductive failure has been established.  In 1990, Stagnaro-Green evaluated a selected obstetric population with a prior history of poor reproductive performance, and was able to show a relationship between antithyroid antibodies and miscarriage.  (1).  This was subsequently confirmed by Glinoer, et al. in 1991 (2).  It was later demonstrated that women who have an increased concentration of antithyroid antibodies and recurrent pregnancy loss do not necessarily demonstrate anticardiolipin antibody (3). Recently, Geva, et al. demonstrated that more than 20% of 78 patients undergoing IVF for mechanical or unexplained infertility tested positive for antithyroid antibodies, and 12% were positive for antiovarian antibodies.  Of note, is the fact that all patients in that study were clinically euthyroid with no history of having been medicated for hypothyroidism (4).  This data suggest that antithyroid antibodies may be independent markers for reproductive failure.

It has been suggested that the existence of antithyroid antibodies, before or during early pregnancy may reflect activated T cell function, which in turn may be related to TH1 lymphocytes (3,5). 

In designing this study, we wished to examine the efficacy of only one variable (the use of IVIg) on outcome in IVF patients who demonstrated thyroid antibodies.  Because of the recent controversy over the use of aspirin and heparin in patients undergoing IVF (8, 9), we elected to treat all patients with aspirin and heparin, thereby eliminating the potential that this variable could have an impact on outcome results when studying the effects of IVIg on these patients.

MATERIALS AND METHODS

PATIENTS

A prospective study was undertaken to evaluate whether reatment with Heparin/Aspirin alone versus combined H/ A + IVIg would influence IVF success rates.

Eighty two (82) women < 40 years of age, who tested positive for ATA, but negative for antiphospholipid antibodies (APA) were randomly placed into two groups in a non-discriminating quasi alternating fashion.  Cases of male infertility, ovum donation, and gestational surrogacy were excluded.  Group A comprised 37 women who received H/A alone while Group B consisted of 45 women who received H/A in combination with intravenous immunoglobulin G (IVIg – Gammimune, Bayer Biological or Venoglobulin, Alpha Therapeutic Corp) 7-14 days prior to embryo transfer.

Patients who had abnormally low plasma levels of IgA were considered to be at risk for the development of anaphylaxis and were selectively medicated with antihistamines and corticosteroids prior to and during the 2-3 hour IVIg infusion.  A second infusion of IVIg was given upon the chemical diagnosis of pregnancy through quantitative serum HCG measurement and a final IVIg infusion was performed upon ultrasound confirmation of a viable pregnancy (between the 6th and 7th gestational week).  All patients underwent controlled ovarian hyperstimulation (COH) using premenstrually administered gonadotropin releasing hormone agonist (lupron-Tapp pharmaceuticals), followed by menotropin therapy, as previously described (7).  The measurement of APA’s was performed as previously described by Matzner, et al. (8).

Antithyroid antibody positivity (ATA+) was defined by the detection of antithyroglobulin and/or antimicrosomal antibodies as measured by the QUANTA Lite Thyroid T and Thyroid M ELISA assay from INOVA Diagnostics (San Diego, CA).  Briefly, 100 microliters of prediluted controls or diluted samples were added to the microwell plates (which were coated with thyroglobulin or microsomal antigen at the factory), and incubated at room temperature for 30 minutes.  The plates were washed in a wash buffer three times, and 100 microliters of HRP Conjugate was added to each well. The plates were then incubated for another 30 minutes.  The plates were again washed three time,s and 100 microliters of TMB Chromogen was added to the wells, and incubated for 30 minutes. At that time, 100 microliters of stopping solution was added, and the absorbance read at 450 nm, using 550 nm as a reference wavelength.  The published relative sensitivity for this assay is 96.8%, and the relative specificity is 94.7%.

DETERMINANTS OF OUTCOME

The number of babies born per transferred embryo, was determined in order to provide a measure of the viable implantation rate.  Multiple births and miscarriages were documented.  A successful IVF outcome was defined as a live birth.

STATISTICAL METHODS

Data was placed into two – by – two Tables: And analysis between and within groups was performed using the Chi Squared Test for significance.  P values below 0.05 were considered to indicate statistical significance.  Analysis was performed using the CHITEST and CHIINV functions for Microsoft Excel 97 for Windows.

Rest of the article can be found at the link provided above. 

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. 


Consulting Website: https://healthcareanalytics.biz

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: Antibodies to phosphatidylethanolamine and phosphatidylserine are associated with increased natural killer cell activity in non-male factor infertility patients

William Matzner, MD
William Matzner, MD, Simi Valley, California

Excerpt: Antibodies to phosphatidylethanolamine and phosphatidylserine are associated with increased natural killer cell activity in non-male factor infertility patients

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

Introduction

Numerous investigators have shown an increased prevalence of antiphospholipid antibodies (APA) among infertile women. However, the exact relationship between APA and infertility in general, and IVF specifically, remains an enigma (Coulam, 1999). Four studies suggest that APA exert an adverse influence on IVF outcome (Birkenfeld et al., 1994; Geva et al., 1994; Sher et al., 1994, 1998a; Dmowski et al., 1995), while five others show no such relationship (Gleicher et al., 1994; Birdsall et al., 1996; Denis et al., 1997; Kowalick et al., 1997; Kutteh, 1997). Possible explanations for the discrepancies include: (i) absence of standardization in the assays used to measure APA; (ii) varying cut-off points used to define positive versus negative results; (iii) differences in the populations of patients studied; and (iv) the fact that IVF, by its very nature, involves so many sensitive and complex steps, as to render assessment of the influence of any single variable on outcome, virtually impossible.
 
The authors have previously reported a correlation between APA positivity and decreased IVF pregnancy rates in cases of organic female and unexplained infertility, which could not be established in cases of isolated male factor infertility (Sher et al., 1994, 1998b). The IVF outcome in these patients was significantly improved through administration of mini-dose heparin/aspirin (H/A) therapy (Sher et al., 1994, 1998b). However we noted that, in contrast to other phospholipid epitopes, in the presence of IgG or IgM class antibodies against phosphatidylethanolamine (PE) and/or phosphatidylserine (PS), H/A therapy alone was not found to be beneficial (Sher et al., 1998b). In these patients, the addition of empiric treatment with intravenous immunoglobulin G (IVIG) was able to improve outcome in a subsequent IVF cycle (Sher et al., 1998a,b). The therapeutic role of IVIG for treating reproductive failure is controversial (Balasch et al., 1996; Christiansen, 1998; Daya et al., 1998; Stephenson et al., 1998). However, proponents of its use for both immunological spontaneous abortion and IVF failure have suggested that a possible mechanism of action may be through down-regulation of NK cell cytotoxicity (activity), thereby converting a hostile Thl endometrial milieu to a trophoblast-friendly Th2 environment (DePlacido et al., 1994).

The present study had two objectives. The first was to evaluate the prevalence of APA and increased peripheral NK cell activity (NKa) in IVF candidates with organic female indications (i.e. endometriosis, pelvic adhesions) or unexplained infertility, compared with a similar group of patients with isolated male factor infertility. Second, given our previous experience of IVIG being beneficial for IVF outcome in aPE/ aPS+ patients, as well as its reported down-regulatory effect on NK cell activity, we attempted to evaluate the association of the presence of antibodies against these specific phospholipid epitopes with increased peripheral NK cell activity.

Materials and methods

Patient population

All patients evaluated between December 1998 and June 1999 for treatment with IVF-embryo transfer who were aged under 40 years, and had cycle day 3 FSH concentrations < 10 mIU/ml, were included in this retrospective analysis. All of these patients were screened for immunological abnormalities as part of a standard work-up. Indications for IVF treatment included male factor, endometriosis, pelvic adhesions and unexplained infertility with previous treatment failure. Patients with a male factor and other female factors were classified in the female factor group. Endometriosis patients encompassed all stages of disease, but were mainly stages I and lI. Patients classified as unexplained infertility were documented by laparoscopy to have patent tubes, were free of pelvic adhesions and endometriosis, and had a normal uterine cavity by hysteroscopy or hysterosalpingography. They had normal ovulation, and there was no evidence of male factor or antisperm antibodies. Not all patients evaluated subsequently underwent treatment and therefore, no attempt was made in this study to correlate the presence of APA or NKa with IVF outcome.

Laboratory evaluation

Assays were performed by Reproductive Immunology Associates (Van Nuys, CA, USA) and by University Health Sciences Laboratory (Chicago, IL, USA). Patients were screened for the presence of antiphospholipid antibodies, using an enzyme-linked immunosorbent assay (ELISA) for IgM, IgG and IgA isotypes to six phospholipid epitopes [cardiolipin (CL), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG) and phosphatidylinositol (PI)], as described previously in detail (Matzner et al., 1994).

The control group for the APA assays consisted of 40 non-infertility patients, aged between 25 and 45 years, who had no history of clinical or subclinical autoimmune disease, or recurrent pregnancy loss. Using the central limit theorem, the sampling distribution of the sample mean was approximated by a normal probability distribution as the sample size became `large' (defined as n > 30). Based upon this theorem, borderline positives were defined as >2 SD above the mean of normal controls, and positive values were defined as >3 SD above the mean for normal controls. As is standard in the field of rheumatology, and as defined by the American Society of Reproductive Immunology (Coulam et al.. 1999), a positive assay in the presence of the proper clinical history was used to define the autoimmune reproductive failure syndrome in these patients.

Each time an ELISA assay was performed, both known negative and positive controls were run simultaneously for each isotype of every epitope. Positive controls were obtained from APL Diagnostics (Louisville, KY, USA), and from serum samples in the radioimmunoassay laboratory that were consistently over 2.0 optical densities (OD). This was important to assess the performance of the antigen coated on each plate, the antibody conjugates, the pipetting technique, the washing method, the incubation times, the incubation temperatures and the substrate. Intra-assay variation was addressed by running each sample in duplicate with the final reported value being the average of the two. The inter-assay assay coefficient of variation was 2.12%.

The determination of natural killer cell function was performed by flow cytometry using a previously described technique (Kane et al., 1996). Briefly, K562 cells were grown as stationary cultures at 37°C in 5% CO2. The cells were subcultured for 3 days before the assay, to be certain that they were in log phase. Before use in the assay, cells were incubated with 10 ill of 30 mmol/1 dioctadecyloxacarbocyanine perchlorate (DiO) per ml for 20 min at 37°C, 5% CO2. Effector cells aPE/aPS and NK cell were isolated from the buffy coat of heparinized blood using the FicolHypaque centrifugation. Target cells at the standard concentration and effector cells at various dilutions (1:1, 1:2, 1:4, 1:8) were added to create effector/target ratios from 50:1 down to 6.25:1. A total of 130 ┬Ál of propidium iodide (PI) was added to the tubes, and the mixture was centrifuged for 30 s at 1000 g in order to pellet target, effector cells and PI. Either interleukin-2 (IL-2) or various concentrations of IVIG were added to the assay. and the mixture was incubated overnight at 37°C, 5% CO2. Data were collected for analysis on the Becton-Dickinson FACScan flow cytometer, using the Consort30 (Becton-Dickinson Immunocytometry systems; BDIS) program and Lysis software (BDIS). The spontaneous lysis was subtracted from the actual lysis for each sample. Based upon the control population (noted above), increased NK activity was defined as > 10% killing, with increased killing activity in the presence of IL-2, and decreased activity of at least 50% from the natural state in the presence of IVIG.

Statistical methods

Analyses of differences within and between groups were performed using the chi-square and Fisher's exact tests for significance where appropriate. A P-value < 0.05 was considered statistically significant.

Results

During the study period, 197 patients were evaluated for the presence of APA and NK cell activity. In total, 89 patients (45%) were positive for APA, and 51 of these (57%) were positive for IgG or IgM antibodies against PEPS. Fifty-four patients (27%) had increased NK cell activity. The mean patient age was 35.7 years. Isolated male factor was seen in 63 patients (32%), endometriosis in 54 (27%) and pelvic adhesions in 55 (28%), while 25 patients (13%) had unexplained infertility. Some 65% (35/54) of patients with endometriosis were APA+, and 44% (24/54) also had increased NK cell activity. Among patients with pelvic adhesions and unexplained infertility, 56% (31/55) and 44% (11/25) were APA+, and 27% (15/55) and 28% (7/25) had increased NK cell activity respectively. Endometriosis was almost twice as likely to be associated with the presence of NK cell activity than with other diagnoses.

Forty-five of the 89 (51%) APA+ patients had increased NK cell activity compared with only 9/108 (8%) patients who tested APA negative (P < 0.0001). Forty of 51 (78%) aPE/aPS+ patients had increased NK cell activity compared with 5/38 (13%) of patients who tested negative for aPE/aPS (P < 0.0001). Some 57% (77/134) of patients with organic female or unexplained infertility were APA+, while only 19% (12/63) of patients with an isolated male factor were APA+ (P < 0.004). In addition, 85% (46/54) of patients with increased NK cell activity had organic female infertility, compared with only 15% (8/54) with a pure male factor (P < 0.002). Some 88% (38/43) of aPE/aPS+, female-factor infertility patients had increased NK cell activity, compared with only 12% (4/34) who tested aPE/aPS negative (P < 0.0001) and 25% (2/8) of aPE/aPS+ patients with an isolated male factor (P < 0.0001). 

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. 


Consulting Website: https://healthcareanalytics.biz
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.

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