H-Y antibody titers are increased in unexplained secondary recurrent miscarriage patients and associated with low male : female ratio in subsequent live births

Abstract

Background: The birth of a boy is significantly more common than a girl prior to secondary recurrent miscarriage (SRM) and is associated with a poorer chance of a subsequent live birth. Children born after SRM are more likely to be girls. High-titer antisera specific for male antigens (H-Y) have been shown to arrest development of male bovine embryos efficiently. We consequently questioned the role of H-Y antibodies in women with SRM.

Methods: Serum samples from patients with unexplained SRM (n = 84), unexplained primary recurrent miscarriage (PRM) (n = 12) and healthy women (n = 37) were obtained. The samples were taken during pregnancy (gestational weeks 4-5) for 77 (80%) of the patients. Enzyme-linked immunosorbent assay was used to detect immunoglobulin G antibodies that specifically recognized any of the five recombinant H-Y proteins (EIF1AY, RPS4Y1, ZFY, DDX3Y and UTY) and their H-X homologs.

Results: H-Y-specific antibodies were more frequent in SRM patients (46%) compared with female controls (19%, P = 0.004) and PRM patients (8%, P = 0.01). The presence of H-Y antibodies in early pregnancy was associated with a low male: female birth ratio among the subsequent live births, as only 12% of children born to H-Y antibody-positive patients were boys compared with 44% boys born to H-Y antibody negative patients (P = 0.03).

Conclusions: The high frequency of H-Y antibody-positive SRM patients and the association between the presence of these antibodies in early pregnancy and the low number of male offspring, suggest that maternal immune responses against H-Y antigens can cause pregnancy losses. Further exploring these mechanisms may increase our understanding of unexplained SRM.

Figure 1

H-Y and H-X antibody responses in serum samples from patients with unexplained recurrent miscarriage and healthy women. Heat maps visualizing the antibody response in OD units to each H-Y and H-X protein of each participating individual, grouped according to patient or control status and sorted with the highest mean OD at the top. Positivity is defined as OD ≥0.1. H-Y-specific responses are responses directed at the H-Y protein and not the corresponding H-X protein. For patients who were pregnant at blood sampling, pregnancy outcome is given. G, girl; B, boy; 0, miscarriage. For control women pregnancy history is given. 2B: given birth to only two boys, 3B: given birth to only three boys, NP, never pregnant.

Figure 2

Representative western blots confirming the ELISA results. Recombinant p24, H-Y and H-X-specific proteins produced in Escherichia coli were probed in western blots with antibodies specific for the V5 epitope tag and serum obtained from patients, as indicated below. The arrow indicates the location of the full-length recombinant H-Y protein. Corresponding ELISA results (in parenthesis) are reported as OD units, determined by absorption at 550–450 nm. 2.1 SRM pregnant patient who gave birth to a girl (UTY 0.55, UTX negative). 2.2 SRM pregnant patient who miscarried (UTY 0.27, UTX negative). 2.3 SRM pregnant patient who miscarried (EIF1AY 0.22, EIF1AX 0.17). 2.4 SRM pregnant patient who gave birth to a girl (EIF1AY 0.22, EIF1AX negative). 2.5 SRM pregnant patient miscarried (RPS4Y 0.13, RPS4X negative). 2.6 Control woman, 2 prior births (boys) (RPS4Y 0.48, RPS4X negative).