April 2000
Volume IV, Number 2
ISSN: 1537-6583
Pages: 090-098

Carolyn B. Coulam, M.D., Cammi Goodman

The Center for Human Reproduction, 750 N. Orleans St., Chicago, IL 60610, Tel: (312)397-8008 Fax: (312)397-8382

Abstract

Intravenous (IV) immunoglobulin (Ig) has been previously shown to increase pregnancy rates after previously failed in vitro fertilization (IVF) embryo (ET) attempts in women who are efficient embryo producers (fertilize at least 50% of oocytes retrieved and generate at least 3 embryos/cycle). Women experiencing implantation failure have a higher frequency of elevated percentage of circulating CD56+ (natural killer) cells (>12%) than fertile women (3-12%). To evaluate the effects of IVIg on pregnancy rates in women with elevated percentage of circulating CD56+ cells, 32 women who had previously failed IVF/ET (>12 embryos transferred without pregnancy) were studied. Pregnancy and live birth rates with and without IVIg were compared in the same woman. All 32 women had previously failed to conceive after at least 12 ET, were efficient embryo producers and had persistently elevated plasma concentrations of CD56+ cells. Each woman received IVIg 500mg/kg prior to ET. If serum hCG concentrations were positive for pregnancy, IVIg was continued at 500mg/kg/mo until 28 weeks gestation. Pregnancy rates with and without IVIg were 56% and 9% (P<0.0001). The rate of live birth was 38% with IVIg and 0% without IVIg (P<0.0001). IVIg enhances pregnancy and live birth rates in women with elevated circulating CD56+ cells who have a history of implantation failure.

Despite technologic advances leading to enhancement of fertilization rates after in vitro fertilization (IVF) (1, 2) implantation rates after embryo transfer (ET) have not increased significantly (3) over the last 20 years (4). Implantation rates after IVF/ET are influenced by the quality of the embryos and receptivity of the endometrium (3-9). Endometrial receptivity involves both hormonal (10-13) and immunologic (14-29) factors. Among the immunologic factors that play a crucial role in successful implantation are natural killer (NK) cells (14-18). NK cells present within the decidua that express CD56(but lack CD 16) have been associated with successful implantation (14-18). A deficiency of decidual CD56+ CD16- cells (18) and an increase in circulating CD56+ cells (25, 26) have been observed in women experiencing implantation failure. Women experiencing implantation failure after IVF and multiple ET have been successfully treated with intravenous (IV) immunoglobulin (Ig) (27). IVIg reduces activation of NK cells and NK killing activity both in vitro (29) and in vivo (30-31). This reduction in activation of NK cells is essential for normal implantation to occur (14). To further define the role of IVIg for treatment of implantation failure, pregnancy and live birth rates were compared before and after IVIg treatment in women undergoing IVF/ET who had elevated levels of circulating CD56+ cells.

Materials and Methods

Patients
A total of 32 women experiencing IVF failure and who had documented elevation of circulating CD56+ cells were offered the opportunity of participating in an Institutional Review Board Approved Clinical Trial using IVIg. A reproductive history was obtained from each of the women and the number of years of infertility and the number of previous clinical ectopic and biochemical pregnancies were recorded. The number of oocytes retrieved and fertilized and of embryos transferred during previous IVF/ET attempts were calculated. An IVF failure was defined as no signs of implantation after transfer of > 12 embryos (95^th percentile for women achieving pregnancy in the IVF/ET program during the study period). To be included in this study each woman had to have had at least 12 embryos previously transferred without signs of implantation and had to have generated at least 3 embryos for transfer each cycle (27). In addition, all women demonstrated elevated levels of circulating CD 56+ cells (>12%) (24) and lack of circulating antiphospholipid antibodies and circulating lupus-like anticoagulant before entry into the study. All women had unexplained IVF/ET failure without obvious ovulatory , male or uterine factors. Women with a history of IgA deficiency or hypersensitivity to IVIg were excluded from the study. Each woman had blood screened for the presence of human immunodeficiency virus and hepatitis C antibodies as well as hepatitis B antigen.

Flow Cytometry Analysis
Ten ml of heparinized blood (green topped tube) was drawn from each woman. The subset of leukocytes expressing CD56+ were identified by flow cytometry and expressed as a percentage of lymphocytes present. Peripheral blood was mixed with FITC monoclonal antibody to CD56 (Ortho Diagnostic, Raritan, NJ). Immunofluorescence analysis of the lymphocyte population was performed on Cytron Absolute Flow cytometer (Ortho Diagnostics, Raritan, NJ). The instrument was aligned and calibrated daily as described by the manufacturer to standardize the fluorescence intensity measurements. Data was displayed on four-decade lot scale. Cells within the lymphocyte cell gate as determined by forward angle light scatter and side angle light scatter were evaluated for fluorescence after reaction with the antibodies. Mouse isotype-matched immunoglobulins as control were run concurrently with lymphocytes from all individuals (32). Normal adult women with no history of recurrent miscarriage 8 to 14 wk pregnant were used as controls for statistical comparison of CD56 lymphocyte populations during pregnancy. There was a 95% confidence range from 2 to 12% with mean of 6.9% when the CD56 levels were determined.

Protocol
Multiple follicular development was induced using the GnRH analogue (GnRHa), leuprolide acetate (LA, Lupron, TAP Pharmaceuticals, Chicago, IL) plus follicle stimulating hormone (FSH, Metrodin, Serono, Inc. Randolph, MA) and was monitored by serial hormone and ultrasonographic measurements (33). Follicles were aspirated under transvaginal ultrasonographic guidance 34 to 36 hours after injection of 10,000 units hCG (Profasi, Serono, Inc, Randolph, MA). All women received IVIg 500mg/kg (Gammagard; Hyland Division, Baxter, Glendale, CA, USA). Gammagard Immune Globulin Intravenous (Human) is produced from Cohn-Oncley fraction II precipitate using an ion-exchange purification procedure and treatment with organic solvent and two detergents prior to embryo transfer. Embryo transfer occurred 48 hours after oocyte retrieval or 24 hours after thawing pronuclear zygotes as previously described (33). The luteal phase was supplemented with progesterone 50-100mg IM in oil each day. If implantation occurred, the woman received IVIg 500 mg/kg every 28 days until delivery or until 28-32 weeks of gestation. The average pregnant woman received 30g IVIg per month for 8 months.

Data Analysis
Comparisons of pregnancy and live birth rates before and after treatment with IVIg were made with Fishers Exact Test. Significant differences were defined as P < 0.05 in a two-tailed test.

Results

The mean age of the women was 37.0 year (range 24 to 42), mean gravidity was 0.3 (range 0-3) and mean number of embryos transferred per cycle was 3.0. The mean percentage of circulating CD56+ was 18% (range 13%-34%).

Pregnancy and live birth rate with and without treatment are shown in Table I. Eighteen (56%) of women with elevated circulating CD56+ cells became pregnant in the IVIg treatment cycle compared to 3 (9%) without IVIg (P < 0.0001). Similarly the live birth rates were significantly higher after IVIg treatment than before (38% vs. 0%) (P = 0.0001).

The outcomes of the 18 pregnancies are summarized in Table II. Twelve women delivered 20 babies including two sets of twins. All infants were born after 36 weeks of gestation and weighed more than 2500 gms. There were no maternal nor fetal complications. Among the 6 pregnancy losses were 3 biochemical pregnancies (2 rising serum hCG concentrations without intrauterine gestational sac demonstrated with transvaginal ultrasound 3 weeks after embryo transfer), 1 ectopic tubal pregnancy and 2 spontaneous abortions. Chromosomal analyses of the abortuses were 94 xxxx + 13 + 13 and 47 xx + 16.

Of the 18 pregnancies, 14 had intrauterine gestational sacs demonstrated on ultrasonographic examination 3 weeks after embryo transfer (5 weeks of gestation). These 14 women had 21 intrauterine sacs seen at 5 weeks gestation. The natural history of these 22 intrauterine gestational sacs is listed in Table III. Eight women had a single intrauterine gestational sac at 5 weeks gestation. One aborted before the sixth week of gestation (karyotype 94 xxxx + 13 + 13) and one aborted at 8 weeks gestation (karyotype 47 xx + 16). The remaining 6 women had singleton term deliveries with no complications. Two gestational sacs at 5 weeks gestation were observed in 5 women. Two women delivered healthy twins at 37 and 38 weeks of gestation. One women had a cesarean section. Both deliveries were otherwise uncomplicated. The remaining 3 women who had 2 intrauterine gestational sacs demonstrated at 5 weeks of gestation had spontaneous deliveries of a single infant. Intrauterine embryo demise of the second twin was diagnosed at 7 weeks, 8 weeks and 8 weeks, respectively. All three intrauterine sacs resporbed spontaneously. One woman implanted 3 embryos visible on ultrasonographic examination at 5 weeks gestation. She delivered a healthy female infant at 37 weeks of gestation without complication. The other 2 embryos had cardiac activity seen at 6 weeks gestation but subsequently had no cardiac activity at 7 and 8 weeks gestation, respectively. Both of these gestational sacs resorbed spontaneously.

Discussion

Analysis of the results indicate that IVIg is useful in the treatment of unexplained IVF failure in women who have elevated circulating CD56+ cells. Women with elevated circulating NK cells have been shown to loose karyotypically normal pregnancies (24). Increased concentrations of activated NK cells have been found in decidual bed biopsies of women with incipient abortions (17). The trophoblast is resistant to killing by CD8+ and CD56+ CD16- NK cells but is susceptible to killing by activated NK cells or LAK cells (34). Thus in order for implantation to be successful, activation of NK to LAK cells must be suppressed. A number of cytokines present in the decidua has been shown to suppress NK activation. These cytokines include a 34 kd protein secreted by activated CD8+ suppresser cells which express progesterone receptor (28), TGF b₂ like substances (18) and TJ6 (35). IVIg has also been shown to suppress NK killing activity both in vitro (29) and in vivo (30, 31). Thus, an attractive explanation for our results is that IVIg down regulated the killing activity of increased natural killer cells enhancing the survival of the implanting blastocysts and consequent live births.

Circulating CD56+ cells serve as a marker to identify women experiencing implantation failure who are likely to respond to IVIg treatment. Still only half of treated women become pregnant. The other half of women who fail to implant could have problems with embryo quality or endometrial receptivity by mechanisms not responsive to IVIg. Other mechanisms that enhance endometrial receptivity included those mediated through hormones (9-13). Hormonal supplementation in luteal phase with progesterone has been shown to enhance implantation and live birth rates in women undergoing assisted reproductive technologies (11). All women in the current study received only progesterone supplementation during the luteal phase. Pregnancy and live birth rates have been significantly increased in some women by adding estradiol to the standard luteal phase supplementation by progesterone (36-38).

The results of this study provide preliminary data and rationale for performing a randomized placebo-controlled clinical trial using IVIg to treat women experiencing IVF failure who demonstrate elevated levels of circulating CD56+ cells. The expected results of the clinical trial can be estimated by the following information in the literature. Approximately 50% of blastocysts available for implantation are chromosomally abnormal (39). After IVF/ET, 15% of preembryos implant (3). Among the 15% that implant, 20% abort (3, 40) and 60% of those that abort are chromosomally abnormal (41). Then it can be calculated that of the 85% of blastocysts which did not implant 37% (50% normal blastocysts available for implantation minus 13% normal blastocysts that implanted) are karyotypically normal. Among the 37% of chromosomally normal preembryos that did not implant two thirds would be expected to have immunologic (22, 26, 27) and one third hormonal (36) mechanisms. Therefore, if IVIg were successful in treating all of the immunologic mechanisms, the pregnancy rate after ET would be expected to rise from 30% (3) per cycle to 56% (30% + 26%) and the live birth rate from 11% (3) per cycle to 37% (11% + 26%). The anticipated pregnancy and live birth rates of 56% and 37% compare closely to the 56% pregnancy and 38% live birth rate obtained after IVIg treatment in the current study.

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Table I

Pregnancy and live birth rates with and without IVIg treatment in women with a history of implantation failure who have elevated circulating CD56+ cells.

Table II

Summary of outcomes of 18 pregnancies occurring in women with elevated levels of circulating CD56+ cells who received IVIg treatment.

Table III

Pregnancy outcome after visualization of intrauterine gestational sac on ultrasonographic examination at 5 weeks gestation.