Middle East Fertility Society Journal, Vol. 12, No. 3, 2007, pp. 188-192

Prediction of pregnancy outcome by limulus amoebocyte lysate (LAL) test in non pregnant women with poor obstetric history

Mohamed Motawe, M.D., Essam Abdullah, M.D.

Department of Obstetrics and Gynecology, Department of Clinical pathology, Suez canal University, Egypt
Correspondence: Mohamed Metawe, M.D, Department of obstetrics and Gynecology, Department of Clinical pathology, Suez canal University, Egypt

Code Number: mf07036

ABSTRACT 

Objective: to evaluate the prognostic value of Limulus Amoebocyte Lysate (LAL) Test in prediction of pregnancy outcome in women with poor obstetric history.
Materials and Methods: non pregnant females with history of abortions and or preterm labor were included. Measurement of bacterial endotoxin in menstrual effluent was done using the Limulus Amoebocyte Lysate (LAL) Test.  Sixty eight women got pregnant within a year; follow up of these women was done to assess the outcome of pregnancy and compared to results of LAL test.
Results: There was a statistically significant correlation between LAL test level and the occurrence of pregnancy (p < 0.05). Spontaneous preterm labor and abortion were 16.6% in samples with endotoxin ≤150 pg/ml while, they were 85.2% with endotoxin >150pg/ml.  Measurement of endotoxin level had a sensitivity of 79.3%, specificity of 89.2% for prediction of occurrence of preterm labor and abortion. It has positive predictive value of 85.2% and negative predictive value of 85.4% and overall accuracy of 85.3%.
Conclusion: Limulus Amoebocyte Lysate (LAL) Test in menstrual blood may be considered as a prognostic marker for pregnancy outcome in women with history of previous abortions and or preterm labor.

Keywords: pregnancy outcome, poor obstetric history, limulus amoebocyte lysate.

Approximately 12% of births are preterm, 70% of these preterm births are due to spontaneous preterm labor with or without prematurely ruptured membranes (1). There is great interest in the role of infection as a primary cause of preterm labor in pregnancies with intact membrane. Some women may have chronic infection even between pregnancies which could cause repeated spontaneous preterm deliveries (2).

Bacterial endotoxin is Lipopolysaccharride component of the cell membrane of gram negative.

The powerful and harmful bacteriological effects of endotoxins result from its ability to induce the production of pro-inflammatory cytokines. The detection of endotoxin in menstrual effluent may indicate microbial invasion of the endometrium and may hinder the implantation and development of the embryo (3).

The Limulus Amebocyte Lysate (LAL) test is rapid, specific, easy to perform and highly sensitive method for the quantitation of Gram negative bacterial endotoxin. It is an aqueous extract of blood cells (amebocytes) from the horse shoe crab. Gram negative bacteria endotoxin (LPS) causes limulus blood to clot. The kinetic turbidimetric method is based on the development of turbidity of the lysate in the presence of endotoxins (4).

Investigators studied the association between bacterial endotoxin in menstrual effluent and pregnancy rate after in vitro fertilization and embryo transfer (IVF-ET) (5), but no study of the association between bacterial endotoxin in menstrual effluent and preterm labor or spontaneous abortion was concluded till now.  In the present study we wished to evaluate the effect of bacterial endotoxin (Gram negative bacteria, Escherichia Coli) in menstrual effluent on spontaneous abortion and preterm labor.

MATERIALS AND METHODS 

The present study was conducted on women attending the Out patient's Clinic of Obstetrics and Gynecologic Department in Suez Canal University Hospital during the period from October 2004 to April 2007.

We included married non pregnant females with history of one or two spontaneous abortions, history of preterm labor not associated with premature rupture of membrane (PROM), no contraceptive method, negative history of anti-phospholipid syndrome, Negative anti-cardiolipin test, Normal blood sugar, and normal kidney function tests. All women gave consent and the ethical committee approved the study.

Exclusion criteria were women with a history suggestive of chromosomal disease or translocation carrier disease of either parent, positive Gram stain for bacterial vaginosis , history suggestive of positive Toxoplasmosis, Rubella , Cytomegalovirus and Herpes (TORCH) disease, Congenital uterine anomalies or uterine masses (Fibroid), Incompetent cervix, hydrosalpinx, Active pelvic inflammatory diseases (PID), Extrauterine infection (Pyelitis-Pneumonia) and  previous caesarean section.

All cases were subjected to Full history taking, full general, abdominal and pelvic examination including speculum examination. Day 21 serum progesterone, ultrasonography, hysterosalpingography, and measurement of bacterial endotoxin in menstrual effluent using the Limulus Amoebocyte Lysate (LAL) Test  Limulus Amoebocyte Lysate test gives positive result at a level >10 pg/ml.

Follow up

Follow up of the women for 18 month for detection of early pregnancy, the outcome of this pregnancy (abortion, preterm, full term pregnancy) according to antenatal program.

Sampling procedure for menstrual effluent

Menstrual fluid, as the endometrial material for endotoxin analysis was taken from the uterine cavity on menstrual days 1 to 3. In the dorsal lithotomy position, women under went cleansing and disinfection of the vulva and vagina. An unmoistened sterilized speculum was inserted into the vagina. A sterile plastic catheter (12-sized) connected to sterile plastic syringe was inserted into the uterine cavity transecervically, and intrauterine aspiration was performed under negative pressure induced by manual suction of a syringe pump. The materials obtained were transferred into heparinized endotoxin-free coded glass containers that were used exclusively for endotoxin assay.

Sample Collection

The materials obtained were transferred into pyrogen free glass coded tubes that were used exclusively for endotoxin assay, containing 20 u/ml blood pyrogen free heparin (Hurly 1994) and immediately centrifuged at room temperature for 10 minutes, the supernatant is separated into another tube, frozen and stored at -45 °C.  The sampling procedures were repeated in some cases with excessive endometrial debris or scanty menstrual blood.

The study population was divided into three groups: 1st group with bacterial endotoxin level of ≤ 150 pg/ml.  2nd group with bacteria endotoxin level of > 150 pg/ml. 3rd group with negative bacterial endotoxin.

Statistical Methods

Data were statistically represented in terms of range mean, standard deviation (±SD), median, and percentages comparison between different groups in the present study was done using analysis of variance (ANOVA), test and post hoe test for determination of

Table 1. Distribution of pregnancy outcome of the studied subjects (n=68)

ANOVA f  value  = 5.252      P < 0.05*
* = Significant difference

the source of variations. Accuracy was represented using the terms sensitivity, specificity and overall accuracy. Correlations between various variables were done using Pearson Correlation Coefficient (r) with graphic representation using linear egression graph. A probability values (P value) less than 0.05 was considered significant .All statistical calculation were done using computer programs Microsoft Excel version 10 and SPSS (Statistical Package for the Social Science) Statistical Program.

RESULTS

The present study include 128 women aged 18-34 years The study group consists of 65 women had a history one or two spontaneous abortion ,59 had a history of preterm labor and 4 women had both history. The mean age of the studied subjects was 27.09 years and 60.3% were housewives. The occupation of our studied subject did not require vigorous physical exertion, prolonged walking or long standing (Table 1). The endotoxin level in menstrual effluent was detectable with a range 5.2 to >1000 pg/ml in 123  samples and was not detectable in 5 samples, with the mean endotoxin level of > 242.88 ± 337.79 pg/ml. 20.3% of women had normal endotoxin level (< 10 pg/ml) (Figure 1).

In our study 68 of the studied subjects became pregnant, 39 (57.1%) women had full term pregnancy, 13 (19.1%) women had preterm labor and 16 (23.5%) of pregnancy ended by spontaneous abortion, the mean endotoxin in menstrual blood was 178.49 ± 256.31pg/ml for pregnant women (n=68). It was 61.14 ± 97.37 pg/ml with full term pregnancies, while in preterm labor it was 268.72 ± 251.35 pg/ml. Regarding to abortions, the mean endotoxin level was 391.21 ± 356.01 pg/ml (Table 2).

There was a statistically significant correlation between endotoxin level and the occurrence of pregnancy (p < 0.05). Spontaneous preterm labor and abortion were 16.6% in samples with endotoxin ≤150 pg/ml while, they were 85.2% with endotoxin >150pg/ml.

Based on our findings, measurement of endotoxin level had a sensitivity of 79.3%, specificity of 89.2% for prediction of occurrence of preterm labor and abortion.

Table 2. Relation between endotoxin level and pregnancy outcome of the studied subjects (n =128)

X² = 26.979            P-value =0.029        ^*Significant  (<0.05)

It has positive predictive value of 85.2% and negative predictive value of 85.4% and overall accuracy of 85.3% and Likelihood ratio of 0.23.

DISCUSSION

Endotoxin is the name given to a group of heat-stable LPS molecules present in the cell walls of gram-negative bacteria that possess toxic effects (6). These toxins, although intimately associated with the bacterial cell membrane, are constantly shed into the environment of the bacterium much like the daily shedding of the superficial layers of the human skin. Many of the biological effects of LPS are mediated by the pro-inflammatory cytokines (such as interleukin (IL)-1, IL-6, tumor necrosis factor (TNF) etc.). Several of these cytokines have been implicated in the delicate immune system balances that exist within the feto–maternal interface (7).

The endotoxin-induced pregnancy loss may be mediated by the release of various pro-inflammatory cytokines/growth factors resulting from the activation of lympho-hematopoetic cells of maternal origin. These factors may alter the normal physiology of the reproductive organs of the mother, and may shift the delicate estrogen/progesterone ratio required for the maintenance of successful pregnancy (8).

Endotoxin-induced pregnancy losses associated with embryonic death, abortion, preterm labor and delivery have been documented in a number of species (9,10). However, the mechanism of LPS-induced abortion is poorly understood.

This study was carried out to assess the relationship between bacterial endotoxin in menstrual effluent and preterm birth and spontaneous abortion in pregnant women. Regarding to preterm birth, there was significant relation between endotoxin level and preterm birth (p<0.05). These results are in accordance with those of Kundston et al. (2003) who reported that nonpregnant women with interpartum chronic endometritis, characterized by plasma cells, were 2.5 times more likely to deliver before 35 weeks in a subsequent pregnancy. Our findings also agree with the findings of other investigators who recovered micro-organisms by trans-abdominal amniocentesis from as many as 20% of women in preterm labor and without overt clinical infection and with intact membranes.

The presence of endotoxin in menstrual effluent may reflect the presence of microbial invasion or colonization in the endometrium. Gram negative bacteria and sub-clinical upper genital tract infection may play a role in spontaneous abortion and preterm labor

In the present study, our findings indicate that women with endotoxin level > 150 pg/ml in menstrual blood may be unlikely to have successful pregnancy. Preterm labor and spontaneous abortion were significantly higher in women with an endotoxin level > 150 pg/ml with an over all accuracy of 85.3%, a sensitivity of 79.3% and the specificity of 89.62%.

The endotoxin levels of >100pg/ml may be of some biological significance, several studies confirmed that bacterial endotoxin present and concentration of >100 pg/ml in culture media affected the development of embryo in human (5).

Seyffarth et al. confirmed that lipopolysaccharides (LPS) is able to induce nitric oxide synthetase expression in intact fetal membranes in vitro if this mechanism occurs in vivo then it could provide one possible pathway by which premature labor could occur in response to intrauterine infection (11).

In conclusion, measurement of endotoxin in menstrual blood may be considered as a prognostic marker for pregnancy outcome in women with history of previous abortion and or preterm.

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