Autoantigens in ovarian autoimmunity associated with unexplained infertility and premature ovarian failure

Abstract

Objective: To identify ovarian autoantigens associated with ovarian autoantibodies.

Design: Hypothesis-generating prospective study.

Setting: Urban infertility referral centers and academic research institution.

Patient(s): Seventy-four patients with infertility, 19 patients with premature ovarian failure (POF), and 16 healthy control women.

Intervention(s): None.

Main outcome measure(s): Identification of autoantigens.

Result(s): To identify major antigens for ovarian autoimmunity, sera from 74 women with unexplained infertility were screened for ovarian autoantibodies (AOAs) by immunoassay and one-dimensional Western blot. The majority of sera had immunoreactions at 50-56 kDa. Six representative positive infertility sera were used to identify antigens between 40 and 60 kD by two-dimensional Western blot and mass spectrometry. Antigens included aldehyde (retinal) dehydrogenases (ALDH1A1, ALDH1A2, and ALDH7A1), protein disulfide isomerase A3, vimentin, α-enolase, phosphoglycerate dehydrogenase, and selenium-binding protein 1 (SBP1). Sixty percent (24 out of 40) of infertility and POF sera were positive for recombinant ALDH1A1, SBP1, or enolase; 80.7% (21 out of 26) of AOA-positive sera had antibodies to one or more of the three antigens, and only 7% (1 out of 14) of AOA-negative sera had antibodies to recombinant proteins.

Conclusion(s): ALDH1A1 and SBP1 are unique to ovarian autoimmunity associated with infertility and POF, and may provide the basis for specific tests to identify patients with ovarian autoimmunity.

Figure 1

(A & B) (A) One-dimensional Western blot showing examples of immunoreactions against human ovarian proteins (250 μg/gel). A negative control serum (Panel A) and examples of positive sera (Panels B-G) are shown.(B) Frequency distribution of the molecular size of immunoreactive bands among positive sera from women with unexplained infertility (n=50/74). The most frequent bands were at 50-56kDa. The data shown were detected using rat ovarian proteins. The frequency distribution was similar for human proteins.

Figure 2

Sera from women with unexplained infertility react with multiple proteins in two-dimensional Western blots. Examples of sera (1:200 dilution) reactions of three different patients (PT 1-3) are shown (Panels A-C). A control incubation with human ovarian protein in which patient sera was omitted (second antibody control) shows no significant reaction (Panel D). Panel A: Upper spot at about 50kDa shows α-enolase (dotted box). Lower spot shows glyceraldehyde-3-phosphate dehydrogenase reaction at 36kDa (solid box). Panel B: Spots at about 50kDa correspond to aldehyde dehydrogenase (dotted oval). Panel C: spots at about 50kDa correspond to Selenium Binding Protein 1 (dotted box).

Figure 3

Immunoassay of patient sera against recombinant SBP1, Enolase and ALDH1A1. The box plot shows the median (horizontal line), data in the 50th percentile (box) and data range (T-bars) of the optical density (OD) values for control sera (Cnt), infertility sera (Inf) and premature ovarian failure (POF) sera for each protein. The OD values differed significantly from controls for infertility (SBP1, p=0.020; enolase, p=0.009; ALDH1A1, p=0.026) and POF (SBP1, p=0.019; enolase, p=0.009; ALDH1A1, p=0.019). There was no significant difference between OD density values for infertility and POF for each protein (p>0.6). Applying the OD cutoff value for a positive antibody result based on the control mean OD (0.37 for SBP; 0.28 for enolase; 0.46 for ALDH1A1), infertility and POF sera were positive for SBP1 (55%), enolase (40%) and ALDH1A1 (52.5%). 80.7% (n=21/26) of sera positive for AOA, but only 7% (1/14) of those originally negative for AOA had autoantibodies to one or more of the three antigens.