Low Plasma MBL Level is a Risk Factor for Recurrent Pregnancy Loss
Conditions: Recurrent Miscarriage, Spontaneous Abortion, Mannose-Binding Lectin Deficiency, Habitual Abortion, Pregnancy Complications, Pregnancy Loss, Recurrent Pregnancy Loss
Sponsor: Aalborg University Hospital

ClinicalTrials.gov ID: NCT04017754 
 
Title: Low plasma MBL level is a risk factor for recurrent pregnancy loss 
 
Date: March 2021 
 
 
Study protocol:  
 
All women admitted to The Center for Recurrent Pregnancy Loss of Western Denmark from 
January 2016 to March 2020 were identified in this combined case-control and cohort study and 
screened for study eligibility. Only patients with a history of ³3 consecutive spontaneous 
pregnancy losses were included. Both biochemical (confirmed by a positive human chorionic 
gonadotropin (hCG) test) and clinical losses (confirmed by an ultrasound examination and/or 
histology of aspirated tissue from the uterus) documented in hospital records were accepted. 
Verified extrauterine pregnancy losses, complete molar pregnancies, and induced abortions of 
social and genetic reasons were not included in the total number of pregnancy losses. Women 
with significant uterine malformations, significant parental chromosomal abnormalities, irregular 
and/or abnormal menstrual cycle length (<22 and >35 days interval), <3 consecutive pregnancy 
losses (n=26), and/or no p-MBL measurement were excluded. 267 women fulfilled all study 
criteria. The RPL patients were followed until birth of a liveborn child after RPL or until March 
2021, whichever came first.  
 
At the first consultation in The Center for Recurrent Pregnancy Loss of Western Denmark, all 
women underwent a routine diagnostic work-up including collection of an obstetric and 
gynecologic history, a routine blood analysis with plasma mannose binding lectin (p-MBL) 

measurement, a uterine hydrosonography, hysteroscopy or hysterosalpingography, and in most 
cases also a parental chromosomal analysis.  
 
According to the individual health profile, the obstetric history, and the results from blood sample, 
treatment options such as vaginal progesterone, oral prednisolone, intravenous immunoglobulin 
(IVIG) infusion, and/or subcutaneous heparin were considered. In patients with an estimated good 
prognosis, only psychological support and close ultrasound scan monitoring (tender loving care) 
was offered. 
 
Retrospectively, data was retrieved from the RPL clinical database of The RPL Center in Aalborg 
University Hospital, Denmark (Data Protection Agency of Region Nordjylland Approval Number 
2018-5) in which relevant data is collected on patients from their first consultation and until their 
contact to the clinic is terminated. Data on perinatal outcomes of all pregnancies before and after 
RPL was collected at the first consultation, from hospital records, and, when needed, completed 
by telephone or e-mail correspondence. This included information about mode of conception and 
delivery, gestational age (GA), birth weight (BW), gender of offspring, volume of peripartum 
hemorrhage, occurrence of preeclampsia, and perinatal complications. GA was divided into 
preterm and very preterm birth defined as birth <37 weeks and <32 weeks of gestation, 
respectively. BW was divided into low and very low BW defined as <2500 g and <1500 g, 
respectively. Peripartum hemorrhage was divided into moderate hemorrhage of 500-999 ml and 
severe hemorrhage of ³1000 ml lost within 24h after birth including both peripartum hemorrhage 
during vaginal delivery and caesarean section. Peripartum hemorrhage was routinely estimated 
and noted by the midwifes in the delivery records generally by weighing absorbent bed sheets. 

Live births refer to children born alive and who were still alive after 1 week. Miscarriages were 
divided into early and late miscarriages defined as <12 weeks and 12-21+6 weeks of gestation, 
respectively. Stillbirth was defined as fetal death ≥22 weeks of gestation and within 1 week after 
delivery. pRPL was defined as ³3 consecutive pregnancy losses while secondary RPL (sRPL) was 
defined as ³3 consecutive pregnancy losses after the birth of a viable child. Whenever the gender 
of the fetus was known, it was recorded.  
 
Blood samples 
Routine blood analysis comprised the following plasma and genetic biomarkers: plasma level of 
MBL, autoantibodies (anti-nuclear antibodies [ANA], anti-ds-DNA-antibodies and anti-thyroid 
peroxidase [TPO] antibodies, lupus anticoagulant [LAC], anti-cardiolipin antibodies [IgG/IgM ACA] 
and anti-b2-glycoprotein-I antibodies [IgG/IgM aGPL-I]), thyroid stimulating hormone (TSH), 
homocysteine, the HLA-DRB1 genotype and hereditary thrombophilic factors (the prothrombin- 
and factor-V Leiden genetic variants, protein S and C, and antithrombin III).  
 
MBL assay 
Plasma MBL was measured using enzyme-linked immunosorbent assay (ELISA). Patient plasma 
was diluted 1:100 in dilution buffer (20 mM Tris + 10 mM CaCl2 + 1 M NaCl + 0,05 % Triton X-100 + 
0,1 % BSA, pH 7,4) and incubated on mannose-coated ELISA microtitre wells (Mannose from 
Saccharomyces cerevisiae SIGMA M-7504). After each step, wells were washed 3 times with 
washing buffer (Tris-buffered saline + 5 mM CaCl2 + 0,05 % Tween 20) and incubated min. 1 hour 
in room temperature. Biotin conjugated monoclonal anti-MBL (HYB 131-01B-0 1 mg/ml, from 
Antibody Shop) was added to the wells. Then, after incubation and washing, wells were incubated 

in alkaline phosphatase conjugated Streptavidin (Dako code D0396) and finally CSPD substrate was 
added (ELISA-LIGHT CSPD/Sapphire-II T1023, from Applied Biosystem). After 20 min. the wells 
were analyzed by a LUMIstar ELISA reader. The analysis used a control patient plasma sample as 
reference, and a highly purified MBL-standard (SER101) was used for calibration, both tested in 
several dilutions. This method did not allow precise measurement of the exact plasma 
concentration of MBL when <100 µg/l and >5000 µg/l; thus, these levels were noted in the data 
collection as a plasma concentration of 100 µg/l and 5000 µg/l, respectively. The cut-off for low p-
MBL used routinely in Danish laboratories is <500 µg/l. 
 
Ethical approval 
Since only data on routine investigations and interventions in the RPL Center is analyzed and 
reported, no approval from the ethics committee was needed.  
 
 
Statistical analysis plan: 
 
Statistical Analysis 
Data was analyzed using Stata/MP 15.0 for Mac, revision 19 June 2017. Level of statistical 
significance was defined as p <0.05. Patients were divided into 3 subgroups according to their p-
MBL levels: low (≤500 µg/l), intermediate (501-3000 µg/l), and high p-MBL group (>3000 µg/l). 
 
Cross-sectional data on p-MBL levels were compared between RPL patients and the MBL reference 
group and analysed using c2 test; first comparing prevalence of ≤500 µg/l and >500 µg/l between 
the two groups and next the same procedure for intermediate and high levels, respectively. The 

relative prevalence of low MBL was compared using prevalence proportion ratio (PPR) with 95% 
confidence intervals (CI).   
A binominal logistic regression analysis was performed to identify the possible prognostic value of 
low and/or high MBL level on the reproductive outcome in first pregnancy after admission which 
was adjusted for confounding variables known to influence on the reproductive outcome. The 
reproductive outcome was defined 1= pregnancy loss and 0= pregnancy>12 weeks or birth in the 
first pregnancy after admission. Patients with no pregnancy after admission, pregnant at the time 
of admission, and pregnant after admission but <12 weeks of gestation at final follow-up were not 
included in the logistic regression analysis of reproductive outcome. p-MBL level was defined as 
1=p-MBL level ≤500 µg/l and 0= p-MBL level >500 µg/l in the analysis. Confounders included in the 
analysis were measured at baseline and involved age (continuous), BMI (continuous), number of 
pregnancy losses before admission (continuous) and exposure to tobacco smoke (1=yes, 0=no).  
A linear regression analysis was performed to analyze the effect of p-MBL level on birth weight 
and GA of first birth before and after RPL; all as continuous variables.  
According to perinatal data on first birth before and after admission, the differences in categorical 
data were determined by c2 test, but in comparisons in which less than 5 numbers were expected 
the Fisher’s exact test was performed. Differences in continuous variables were determined by the 
two-sample Mann-Whitney U test if data was non-normally distributed, and by t-test if data fitted 
a normal distribution. When analyzing the previous children´s sex, only women with one or more 
children of same sex born before the RPL diagnosis with a GA >22 weeks were included. Women 
with sRPL with ≥2 children of mixed gender were excluded from analyses involving sex of 
child(ren) born before admission. Sex ratio was reported as a male:female ratio.