Shagufta A. Khan

Multiple endometrial antigens are targeted in autoimmune endometriosis

Endometriosis is defined as the growth of endometrial glands and stroma in ectopic locations. Its aetiology is multifactorial, but autoimmunity has been shown to play a role in its onset and development. The present study aimed to investigate the presence of both IgG and IgM anti-endometrial antibodies in sera of endometriosis patients in comparison with age-matched controls, and to also investigate the cognate endometrial proteins involved. Sera from these groups were screened by western blot and immunohistochemistry. Thirteen out of the 40 sera tested were positive for IgG isotype, and 10/27 IgG negative patients were positive for IgM isotype. These findings indicate that endometrial antibodies of IgG and IgM classes could be detected in almost 60% of endometriosis patients. Of the various identified endometrial antigens, 30 and 45 kDa antigens were immunodominant in both IgG and IgM positive endometriosis patients. With immunohistochemistry, positive sera showed reactivity in luminal epithelium, glandular epithelium and stroma. These anti-endometrial antibodies might be partially responsible for failure of implantation leading to infertility. Identification of specific targets would be a help in understanding the pathophysiology of endometriosis, and would also help in setting up a non-invasive test for the diagnosis of endometriosis.

Identification of novel immunodominant epididymal sperm proteins using combinatorial approach

Functionally immature spermatozoa leave the testis mature during epididymal transit. This process of maturation involves either addition of new proteins or modification of existing proteins onto the sperm domains that are responsible for domain-specific functions. Epididymal proteins are preferred targets for immunocontraception. In an attempt to identify epididymis-specific sperm proteins, we used a novel combinatorial approach comprising subtractive immunization (SI) followed by proteomics. Following SI, sera of mice were used for immunoproteomics, which led to the identification of 30 proteins, of which four proteins namely sperm head protein 1, sperm flagella protein 2 (SFP2), SFP3, and SFP4 are being reported for the first time on sperm. Another group of four proteins namely collagen alpha-2 (I) chain precursor, homeodomain-interacting protein kinase 1, GTP-binding protein Rab1, and ubiquinol cytochrome c reductase core protein II although reported earlier in testis are being reported for the first time in epididymal sperm. Furthermore, seven out of these eight novel proteins could be validated using peptide ELISA. These data are a useful repository, which could be exploited to develop targets for post-testicular immunocontraception or biomarkers for infertility diagnosis and management.

Differential Proteomics Leads to Identification of Domain‐Specific Epididymal Sperm Proteins

The alteration in the protein signatures of the testicular sperm during its epididymal sojourn makes it functionally competent for successful fertilization. The present study was undertaken to identify the proteins acquired on its 2 domains, that is, the head and the flagellum, during the epididymal transit using a differential proteomics approach. Testicular sperm proteome was compared with cauda epididymal sperm proteome in rat. The protein spots exclusively present in the cauda epididymal sperm proteome were searched in the cauda sperm head proteome and the cauda sperm flagella proteome, and a total of 335 spots were found by alignment and auto-matching of the gels, of which 140 could be identified by mass spectrometry. Database search revealed that of these 9 proteins were novels. Gene Ontology annotation revealed that the identified proteins were distributed across different cellular components and were primarily involved in metabolic processes. The study also provides information on the localization of these proteins on the sperm domains, which indirectly gives a clue about its putative function. Validation of 3 proteins, namely MMSDH, NDUFS1, and UQCRC2, using antibodies very elegantly demonstrates that the strategy has been very effective. This comprehensive data of domain-specific epididymal sperm proteins will be useful in development of newer targets for posttesticular contraception and diagnostic markers for infertility.

Naturally occurring anti-albumin antibodies are responsible for false positivity in diagnosis of autoimmune premature ovarian failure.

Autoimmunity is a well-established causative factor of premature ovarian failure (POF), and evidence for the same has been well reported in the literature. Detection of specific autoantibodies remains the most practical clinical research marker of any autoimmune disease. Variation in efficiency and specificity in the detection of ovarian autoantibodies has been reported. However, the frequency of false positivity and a solution to overcome this has not yet been reported. Herein, we report autoantibody to albumin as the likely responsible agent for false positivity. Our data indicate that presence of naturally existing autoalbumin antibodies in the circulation of normal women is responsible for the false signal seen in SDS-PAGE Western blot analysis and in immunohistochemistry (IHC). Having shown the presence of anti-albumin antibody in normal women as well as in the sera of POF patients, we have developed a novel blocking agent to overcome this problem. A high titer polyclonal antibody against human serum albumin was generated. This antibody showed immunoreactivity to albumin obtained from various sources. Preincubation of Western blots and IHC sections with this antibody drastically reduced background signals. The advantage of using this blocking was evident by identification of specific anti-ovarian antibodies in a group of POF patients. This blocking procedure made it possible to obtain a clear indication of the ovarian antibody status in women presenting with autoimmune POF.

Identification and validation of novel serum markers for early diagnosis of endometriosis

BACKGROUND Non-invasive diagnosis of endometriosis is urgently required to prevent the long delay between the onset of symptoms and diagnosis. A biomarker that possesses both high sensitivity and specificity is greatly required. Here, we describe the use of a proteomic approach to identify potential novel endometrial antigens using sera from endometriosis patients and healthy controls, with evaluation of biomarkers for non-invasive diagnosis of endometriosis. METHODS A cross-sectional study was conducted to identify specific endometrial antigens using 1D and 2D western blots in women with early endometriosis (n = 17), advanced endometriosis (n = 23) and without endometriosis (n = 30). Five immunoreactive spots were analyzed using matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry with MASCOT analysis. ELISAs were established for specific epitopes and autoantibody titres were estimated in an independent cohort comprising women with early endometriosis (n = 18), advanced endometriosis (n = 32) and without endometriosis (n = 27) for validation. RESULTS The 2D western blot analysis resulted in the identification of three endometrial antigens, tropomyosin 3 (TPM3), stomatin-like protein 2 (SLP2) and tropomodulin 3 (TMOD3). Serum levels of antibodies against the epitopes from the immunodominant region of proteins TPM3, SLP2 and TMOD3 were significantly elevated in endometriosis patients when compared with controls. Sensitivity and specificity of serum anti-TPM3a-autoAb (61%, 93%), anti-TPM3c-autoAb (44%, 93%), anti-TPM3d-autoAb (78%, 89%), anti-SLP2a-autoAb (50%, 96%), anti-SLP2c-autoAb (61%, 93%), anti-TMOD3b-autoAb (61%, 96%), serum anti-TMOD3c-autoAb (78%, 93%) and anti-TMOD3d-autoAb (78%, 96%) were better than those of serum CA125 levels (21%, 89%) in the detection of early stages of endometriosis. CONCLUSIONS Serum anti-TPM3a-autoAb, anti-TPM3c-autoAb, anti-TPM3d-autoAb, anti-SLP2a-autoAb, anti-SLP2c-autoAb, anti-TMOD3b-autoAb, anti-TMOD3c-autoAb and anti-TMOD3d-autoAb could be new markers for the early diagnosis of endometriosis.

Evaluation of Contraceptive Potential of a Novel Epididymal Sperm Protein SFP2 in a Mouse Model

Citation Khan SA, Jadhav SV, Suryawanshi AR, Bhonde GS, Gajbhiye RK, Khole VV. Evaluation of contraceptive potential of a novel epididymal sperm protein SFP2 in a mouse model. Am J Reprod Immunol 2011; 66: 185–198

Regulation of acrosome reaction by Liprin α3, LAR and its ligands in mouse spermatozoa

Zona pellucida‐based induction of acrosome reaction (AR) is a popular and well‐accepted hypothesis. However, this hypothesis is being challenged in recent years and it has been proposed that the cumulus cells might be the site of AR. In our previous study, we reported the presence of a synaptic protein Liprin α3 on sperm acrosome, and proposed its role in AR. This study was designed to understand the role of Liprin α3 and its interacting proteins in regulation of AR. It is observed that the presence of anti‐Liprin α3 antibody inhibits the process of AR. Colocalization experiments demonstrate the coexistence of leucocyte antigen related (LAR) protein, Rab‐interacting molecule (RIM) and Liprin α3 on sperm acrosome thereby completing the identification of all the members of RIM/MUNC/Rab3A/liprinα complex required for membrane fusion. This study demonstrates the effect of LAR ligands such as Syndecans, Nidogens and LAR wedge domain peptide on AR. We could see an increase in AR in presence of these ligands. On the basis of these data, we speculate that in presence of ligands or wedge peptide, LAR undergoes dimerization leading to inhibition of phosphatase activity and increase in AR. The presence of one of the ligands Syndecan‐1 on cumulus cells led us to hypothesize that it is Syndecan which induces AR in vivo and thus another site of AR could lie in cumulus.

Immunization with ovarian autoantigens leads to reduced fertility in mice following follicular dysfunction

Immunoproteomics using sera of women with ovarian autoimmune diseases such as primary ovarian insufficiency and IVF embryo transfer recruits led to identification of three proteins namely alpha actinin 4 (α-ACTN4), heat-shock 70 protein 5 (HSPA5), and actin beta (ACTB). This study deals with the establishment of a peptide ELISA for screening sera of antiovarian antibody (AOA)-positive patients and further delves into understanding the role of these three proteins in ovarian autoimmunity in a mouse model. Using in silico approach, antigenic peptides of these proteins were identified and used for peptide ELISA. ELISA results indicated that AOA-positive sera showed reactivity with only specific peptides. The functional significance of the dominant peptides was studied by active immunization of female mice with these peptides. All immunized mice generated high antibody titers and profound effect on ovaries with few primordial (2.4±0.1, 2.4±0.2, and 2±0.1), primary (2.4±0.5, 1.7±0.3, and 2.4±0.3), preantral (2.3±0.5, 3.4±0.3, and 2.9±0.3), antral (0.9±0.2, 1.6±0.8, and 2.3±0.6) follicles, and corpora lutea (2.8±0.8, 2.9±1.7, and 4.6±2.3), and increased number of atretic follicles (5.5±0.4, 4.9±1.8, and 7.5±1.0) in ACTN4-, HSPA5-, and ACTB-immunized mice compared with control animals (3.0±0.2, 3.5±0.6, 3±0.1, 3.6±0.2, 4.7±0.3, and 1.5±0.3) respectively. These mice when mated with fertile male mice showed an overall 25-43% reduction in fertility compared with controls. The data clearly suggest that the dominant antigenic epitopes of the three proteins play critical role in fertility and could possibly be the key autoimmune targets. These epitopes could be used to develop a more specific and sensitive diagnostic test for women with ovarian autoimmune diseases and to design therapy for disease management for reinstatement of ovarian function.

Panel of Autoimmune Markers for Noninvasive Diagnosis of Minimal–Mild Endometriosis A Multicenter Study

Endometriosis, characterized by the presence of endometrial-like tissue at extrauterine sites, is a common, chronic, estrogen-dependent, inflammatory condition associated with pelvic pain, subfertility, dysmenorrhea, and dyspareunia, affecting about 10% of reproductive-age women in any population. The diagnosis of endometriosis is usually delayed on an average by 8 to 11 years leading to significant consequences in terms of disease progression. The current study was aimed to validate enzyme-linked immunosorbent assay based on the epitopes of stomatin-like protein 2, tropomodulin 3 (TMOD3), and tropomyosin 3 (TPM3) for diagnosis of minimal–mild endometriosis (revised American Fertility Society Classification (rAFS) stage I-II) and to compare the performance with the reported markers: cancer antigen (CA) 125, CA19-9, α-enolase, Serine/threonine-protein kinase (PDIK1L), and syntaxin 5. This was a cross-sectional, multicenter study conducted during the year 2012 to 2015. Women with minimal–mild endometriosis (rAFS stage I-II [n = 133]) and healthy controls (n = 104) were screened for 11 novel autoimmune markers and reported markers α-enolase, PDIK1L, syntaxin 5, CA-125, and CA19-9. The sensitivity and diagnostic accuracy of serum antibodies against all the 11 epitopes were higher than that of CA-125, CA19-9, α-enolase, PDIK1L, and syntaxin 5 for diagnosis of rAFS stage I to II endometriosis. The sensitivity of 6 biomarkers (anti-TMOD3b-autoAb, anti-TMOD3c-autoAb, anti-TMOD3d-autoAb, anti-TPM3a-autoAb, anti-TPM3c-autoAb, and anti-TPM3d-autoAb) was higher at the specificity of ≥80% for diagnosis of rAFS stage I to II endometriosis as well as ultrasound-negative endometriosis. Further, logistic regression models of this panel of biomarkers showed increase in sensitivity, specificity, and diagnostic accuracy than individual biomarkers. The panel of 6 autoimmune biomarkers could be useful in setting up of noninvasive diagnostic test for detection of minimal–mild endometriosis.

The CFTR gene mild variants poly‐T, TG repeats and M470V detection in Indian men with congenital bilateral absence of vas deferens

The aim of the study was to detect the frequency of the CFTR gene variants poly‐T, TG repeats and c.1408A>G p.Met470Val (M470V) in Indian men with congenital bilateral absence of the vas deferens (CBAVD). Men diagnosed with CBAVD (n = 76), their female partners (n = 76) and healthy men from general population (n = 50) were recruited. Genomic DNA was isolated and the polymorphic regions of IVS9‐ c.1210‐12T [5] and M470V were amplified using specific primers followed by Sangers DNA sequencing. A statistically significant increase in the frequency of heterozygous IVS9‐ c.1210‐12T [5] (39.4%) was observed in CBAVD men as compared to controls (14%). The allelic distribution of c.1210‐12T [5], c.1210‐12T [7] and c.1210‐12T [9] in CBAVD men was 21%, 64.4% and 13% and that in healthy controls was 7%, 73% and 20% respectively. Longest TG repeat c.1210‐34TG [13] was found in association with c.1210‐12T [5] with an allelic frequency of 5.9% in CBAVD men. We found a significant association of c.1210‐34TG [12]/c.1210‐34TG [13] ‐ c.1210‐12[5] –V470 allele in CBAVD men. Twelve female partners harboured a heterozygous c.1210‐12T [5] allele. The study emphasises the need to screen both partners for the polymorphisms M470V, poly‐T, TG tract repeats in addition to population‐specific known CFTR gene mutations.