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.

Protein repertoire of human uterine fluid during the mid-secretory phase of the menstrual cycle.

BACKGROUND: This study is an attempt to construct a repository of polypeptide species in human uterine fluid during the mid-secretory phase of menstrual cycle. This information is essential to generate alternative and less invasive tools for the assessment of uterine functions. METHODS: Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometric analysis were used to resolve and identify the major components of human uterine fluid. RESULTS: Uterine fluid collected during the mid-secretory phase (n = 6) demonstrated ca. 590 polypeptide spots in the linear range of pH 4-7 after 2D PAGE. Mass spectrometric analysis revealed the presence of heavy and light chains of immunoglobulins, alpha-1 anti-trypsin precursor, anti-chymotrypsin precursor, haptoglobin, apolipoprotein A4, apolipoprotein A1 fragment, beta-actin fragment, heat shock protein 27, hemopexin precursor and transferrin precursor. 2D protein profile of fluid collected during the proliferative phase (n = 5) revealed ca. 433 polypeptide spots, of which 279 could be paired with mid-secretory phase protein spots on the basis of their coordinates (isoelectric point and molecular weight) in 2D gels. Apolipoprotein A4, apolipoprotein A1 fragment and alpha-1 anti-trypsin precursor were 2-3-fold more abundant in uterine fluid collected during the mid-secretory phase as compared with that in the proliferative phase. Further, 86 uterine fluid polypeptides were conserved across species, being detected in human, rat and bonnet monkeys. CONCLUSIONS: The molecular repertoire of the mid-secretory phase human uterine fluid, when compared with that of the proliferative phase uterine fluid, is broadened due to differential expression of proteins. Further, some of the mid-secretory phase proteins were conserved across species.

Dyslipidemia with particular regard to apolipoprotein profile in association with polycystic ovary syndrome: a study among Indian women.

OBJECTIVE: To investigate putative dyslipidemia in women with polycystic ovary syndrome (PCOS) with particular emphasis on specific parameters of atherosclerotic risk, and to assess the independent influence of obesity and hyperandrogenemia on these parameters. SUBJECTS: Women investigated were among those attending the infertility clinics. Three groups of women were studied: (I) a core study group consisting of oligomenorrhoeic, chronically anovulatory women with or without obesity in whom polycystic ovaries were confirmed through ultrasound evidence, and were established to be the cause of infertility; II) a control group of non-hirsute, non-obese women with regular menstrual cycles; and (III) a group of controls consisting of women with obesity, but with normal ovarian morphology and regular menstrual cycles. METHODS: All three study groups were investigated for androgen (total testosterone) and lipid-lipoprotein profile, including apolipoproteins, ApoA1 and ApoB. Retrospective analysis of the data was carried out to assess hyperandrogenism in the study group of women with PCOS as well as to investigate changes in the lipid-lipoprotein profile, particularly the measures of cardiovascular risk, ApoA1 and ApoB. RESULTS: Triglycerides showed a significant increase in the PCOS group compared to controls, whereas HDL-cholesterol as well as HDL-carrying ApoA1 showed a significant decrease (P < .05). Also a significant finding was the decrease in ApoA1/ApoB ratio among the women with PCOS compared to both controls and obese women. A direct negative correlation of this decrease in ApoA1/ApoB ratio with the Body Mass Index was also confirmed in the study. Hyperandrogenemia in terms of significantly raised total testosterone levels was found in 30% of the PCOS women. However, no direct correlation of this increase with changes in lipid-lipoprotein profile could be observed. CONCLUSION: The study confirms the trend toward dyslipidemia among women with PCOS, particularly in parameters associated with cardiovascular risk. A significant association of obesity rather than raised testosterone with this dyslipidemia was also confirmed by the study.

Expression of integrin receptors on peripheral lymphocytes: correlation with endometrial receptivity.

PROBLEM: To investigate the expression of integrin (ITG) cell adhesion molecules on peripheral blood lymphocytes (PBL) and their correlation with endometrial cell ITG expression in fertile and infertile women during peak uterine receptive period (day 19/20). METHOD OF STUDY: Surface marker expression and quantification of alpha6, alpha4 and beta3 ITG subunits was done by immunohistochemistry, indirect immunofluroscence and cell-enzyme-linked immunosorbent assay methods using endometrial cells and PBL obtained from fertile and infertile (unexplained infertility) women. RESULTS: The expression of ITGs was significantly (P<0.001) decreased in the endometrial cells of infertile women compared to normal fertile women. These results correlated well with the data obtained using PBL-ITG expression. CONCLUSIONS: If these preliminary data are consistent in a larger group of patients, the expression of alpha4 and beta3-ITG subunits on PBL may be used as clinical markers to assess endometrial receptivity in infertile women. Moreover, frequent blood sampling is advantageous over repeated endometrial biopsies as the former approach is easier, non-traumatic and avoids intra-uterine infections.

Chlamydia trachomatis infection among asymptomatic males in an infertility clinic.

Chlamydia trachomatis can lead to a variety of complications including tubal infertility. Similarly asymptomatic infection in male partner can also hinder conception. The prevalence of this infection among the infertile female in the Institute's Infertility Clinic was observed to be 34%. Hence the present study was undertaken to find out these infection among the asymptomatic male partners of these infected women. Fifteen asymptomatic males who were not treated with any antibiotics in recent past were enrolled. First voided urine, semen and blood were collected from each individual for diagnosis of this infection. Chlamydia antigen was detected in 33.3% while Chlamydia antibody was detected in seven (46.7%) of these cases. Of these seven, three cases were positive for antigen. This preliminary observation suggests that amongst the infertile couple a sizable percentage (60%) of asymptomatic male partners remain infected with Chlamydia trachomatis.

Poor quality of sperm as it affects repeated early pregnancy loss.

A study was carried out to determine whether males contribute to repeated early pregnancy loss. Semen samples were analyzed from proven-fertile men (n = 51 group I) and from men whose partners presented with early pregnancy loss (>3 first trimester abortions, n = 32 group II). Routine analysis, sperm function tests, and ultrastructural studies of sperms were carried out. Female factor could be identified in 25 (78%) couples, and in 7 (22%) no cause either male or female could be identified and the semen analysis was normal. Percent morphologically normal did not differ significantly between the groups, but increased sperm head abnormalities were seen. The functional tests were all normal except for a significant decrease in the capacity of nuclear chromatin to decondense in vitro. The ultrastructural studies showed defects of chromatin condensation and irregular nuclei with vacuoles. This study points to the loss of chromatin integrity as a possible contributing factor from males to early pregnancy loss.

Integrin cell adhesion molecules in endometrium of fertile and infertile women throughout menstrual cycle.

Integrins (ITGs) are ubiquitous cell adhesion molecules that undergo dynamic alterations during the normal menstrual cycle in human endometrium. The distribution of four different subunits, viz. alpha 4, alpha 6, beta 3 and beta 4 in human endometrial tissue at different stages of the menstrual cycle was studied using immunohistochemical, enzyme immunoassay and SDS-PAGE/Western blot techniques. The specificity of each mAb to their respective ligands viz., laminin (Ln), fibronectin (Fn) and vitronectin (Vn) was done by cell adhesion assays. Both alpha 6 and beta 4 subunits (Ln receptors) expressed primarily on the glandular epithelium, while glandular, stromal and luminal cells expressed predominantly alpha 4 (Fn receptor) and beta 3 (Vn receptor). The appearance of alpha 4 and beta 3 ITG subunits was found to be cell and cycle specific. The levels of both alpha 6 and beta 4 increased throughout the menstrual cycle, while beta 3 subunit appeared abruptly on cycle day 19/20. The immunostaining for alpha 4 and beta 3 was absent in 90% of infertile women. The timing of expression of alpha 4 and beta 3, the two cycle--dependent ITGs framed the putative window of implantation and suggests a role in the diagnosis of infertility. In conclusion, the absence of alpha 4 and beta 3 ITG expression in the endometrium of infertility subjects during mid luteal phase may be associated with defects in uterine function. The defective uterine receptivity may be an unrecognised cause of infertility in these group of women.

Integrin cell adhesion molecules on human spermatozoa.

The expression of integrin cell adhesion molecules (ITG-CAMs) by human ejaculated spermatozoa (fresh, capacitated and acrosome reacted) was evaluated by immunocytochemical, immunofluorescence and cell-ELIS methods, using monoclonal antibodies against alpha-6 and beta-3 subunits. Both the subunits were expressed on the acrosome region in fresh spermatozoa and post acrosomal region after acrosome reaction induced by calcium ionophore. The spermatozoa of the fertile men showed significantly (P < 0.001) higher expression of alpha-6 and beta-3 ITG subunits than the subfertile men. The percentage of spermatozoa reacting with alpha-6 and beta-3 mAbs increased significantly after the loss of acrosome when compared with fresh spermatozoa. Moreover, 35-40% of spermatozoa with normal shape and none of the spermatozoa with pathological shape showed a positive reaction. The quantitative analysis carried out by ELISA suggests that the levels of these ITG subunits decreased significantly (P < 0.001) in the subfertile subjects when compared with the fertile and the difference was more for alpha-6 than the beta-3. Hence our result suggests that alpha-6 subunit may be used as a clinical marker to evaluate the sperm quality in men.

Resazurin reduction test to evaluate semen quality.

The resazurin test (REZ TEST) was performed on semen samples obtained from 75 untreated subfertile and 10 pregnancy confirmed fertile males. The results were compared with other sperm parameters to assess the quality of semen. The highest correlation of REZ grade was with motile sperm (r = 0.90, P < 0.001), sperm viability (r = 0.80, P < 0.001) and sperm concentration (r = 0.71, P < 0.001). The REZ grade indicated significant difference in the rate of positive tests between samples from fertile males and patients exhibiting abnormal semen parameters. The RES TEST had a positive predictive value of 93.75% for a progressives motile sperm concentration of > 20 x 106/ml and a negative predictive power of 88.7% for the same variable at a criterion value of 10 x 106/ml. It is suggested that REZ TEST can be performed with a relatively small volume of semen, it is easy to perform and requires no technical equipments.

Spermicidal activity of Magainins: in vitro and in vivo studies.

Spermicidal efficacy of two synthetic antimicrobial peptides, viz. Magainin-A (Mag-A) and Magainin-G (Mag-G), have been studied under in vitro condition using rat and human spermatozoa. The data showed that sperm motility was inhibited with various concentrations of Magainins at different intervals ranging from 20 to 480 seconds as compared to controls. The minimum concentration of Mag-A required to immobilize spermatozoa (40-60 x 10(6)/ml of saline) within 20 seconds was found to be 100 micrograms in rat and 200 micrograms in human. The action of Mags on sperm motility was observed to be dose-dependent. The activity of Mag-A was significantly (P < 0.05) higher than Mag-G (P < 0.05). The in vivo studies in rats indicated that Mag-A when applied intravaginally, on the day of estrus, at the dose level of 200 micrograms, for five consecutive estrous cycles, induced no abnormalities either in the length of estrous cycle or in morphology of vaginal epithelial cells. When Mag-A was applied intravaginally before mating on the proestrusestrus day, complete arrest of sperm motility with 200 micrograms concentration was observed and none of these treated females conceived after the mating. In view of these facts, it is suggested that Mag-A, having anti-bacterial, anti-viral, anti-fungal and spermicidal activities, could be a potent vaginal contraceptive.

Lymphocytes--their possible endocrine role in the regulation of fertility.

PROBLEM: Peptide secretion by peripheral lymphocytes obtained from fertile and infertile women. METHOD: LH, FSH, and prolactin were estimated in the serum and culture supernatants obtained from Con-A stimulated human lymphocytes by RIA. RESULTS: The levels of LH, FSH and prolactin were low in the culture supernatants obtained from lymphocytes of infertile women, when compared with proven fertile women. CONCLUSION: The results indicated impairment in endocrine functions of lymphocytes obtained from infertile women. This impairment in lymphocyte function could be one of the causes of infertility in these women.

Early pregnancy factor (EPF) as a marker for detecting subclinical embryonic loss in clomiphene citrate-treated women.

PROBLEM: A discrepancy exists between the apparently normal ovulation and the pregnancy rates in women treated with clomiphene citrate (CC). Our previous studies have indicated that immuno-suppressive "early pregnancy factor" (EPF) is a novel marker to detect subclinical embryonic loss in infertile women. METHOD: In the present study EPF was used as a marker to detect subclinical embryonic loss in women treated with CC with/without gonadotropins. In some of the women treated with CC, conception was assisted by artificial insemination with husband's semen (AIH). RESULTS: Our results have indicated that fertilization occurred (EPF + ve) in 47.7% (52/109) of women treated with CC with/without gonadotropins; 13.46% (7/52) retained the fetus and continued pregnancy till full term, whereas 78.9% (41/52) did not retain the fetuses. In the group where after stimulation, conception was assisted by AIH, fertilization was observed in 38.24% (26/68), retention in 11.54% (3/26) but subclinical embryonic loss was observed in 80.8% (21/26) cases. CONCLUSION: Thus, our results have indicated that subclinical embryonic loss may account for some of the discrepancy observed between the apparently normal ovulation and the pregnancy rates in women treated with clomiphene citrate.

Lipoprotein lipids in androgen excess--a study among Indian women.

OBJECTIVE: To assess changes in lipoprotein-lipid profile vis-a-vis putative risk of atherosclerotic diseases, among a group of hyperandrogenic women. SUBJECTS AND METHODS: Women studied were among those being screened for polycystic ovarian disease (PCOD) at the Reproductive Endocrinology Clinic of the institute. They all had oligomenorrhea/chronic anovulation as their problem and had clinical or laboratory evidence of hyperandrogenism, viz., hirsutism or elevated serum testosterone levels. Lipoprotein-lipid profiles obtained in 51 such women were compared with those of 11 controls with normal menstrual cycles and no evidence of hyperandrogenism. RESULTS: No significant changes were observed in any of the lipoprotein-lipid variables which could be attributed to hyperandrogenism per se. However, a combined effect of obesity and raised circulating androgens in causing a significant change in the lipoprotein-lipid profile (increased cholesterol, LDL-C/HDL-C, and cholesterol/HDL-C) was clearly discernible from the study. CONCLUSIONS: Hyperandrogenism by itself may not signal a risk for atherosclerotic diseases. However, the potential of such risk would be significantly enhanced if obesity is associated with high circulating levels of androgens.

Capacitation and the acrosome reaction in sperm from men with various semen profiles monitored by a chlortetracycline fluorescence assay.

Sperm obtained from groups of men with various semen profiles were incubated for 8 h in BWW medium containing human serum albumin to promote capacitation. Capacitation and the acrosome reaction were monitored by a chlortetracycline (CTC) fluorescence assay. Four distinct CTC patterns were observed on the sperm head. No significant difference was observed in the time-course curve of these CTC patterns in sperm obtained from normozoospermic, asthenozoospermic and oligozoospermic men. Spontaneous and A23187-induced acrosome reactions were also comparable in these groups. However, in sperm obtained from teratozoospermic and polyzoospermic men, the increase in CTC pattern associated with capacitation appeared slower and sluggish. In these two groups, the induced acrosome reaction was also significantly lower when compared to that in the other three groups of men. In polyzoospermia, the spontaneous acrosome reaction was significantly lower when compared to all the other groups. Fresh sperm would not undergo the acrosome reaction following A23187 treatment. The results of this study indicate sluggish (defective) capacitation and inability of capacitated sperm to undergo induced acrosome reaction in teratozoospermic and polyzoospermic men as evaluated by the CTC method.

Early pregnancy factor (EPF) as a marker for the diagnosis of subclinical embryonic loss.

The validation of EPF as a possible correlate of early fertilization has made it possible to study and detect fertilization of the ovum in normal fertile women (during the luteal phase) and also in women with infertility, where the fertilization of the ovum may not be affected but there may be impairment in early embryonic development which results in early embryo loss or subclinical embryo loss. Our results have suggested that using EPF as a marker, we could detect subclinical embryonic loss in 57.8% of the infertile women where more than one menstrual cycle was studied and the blood was collected 4-7 days after ovulation. After the missed period, 80% of the patients who were negative for EPF but positive for hCG had spontaneous abortions. It would be interesting to study how EPF behaves as a marker, to detect subclinical embryonic loss in diverse pathological situations such as recurrent abortions, parental age and translocation carrier parents.

Screening infertile women for the assessment of corpus luteal function and their response to therapy by ELISA of pregnanediol glucuronide.

Corpus luteal function was assessed by estimating pregnanediol 3-alpha-glucuronide (PdG) in three midluteal-phase urine samples collected from 85 women attending the infertility clinic. The previously established cut off limits based on PdG estimations were useful in detecting anovulation in 23 cases, corpus luteal adequacy in 42 cases and corpus luteum deficiency (CLD) in 20 cases. In 8 women CLD could be corrected with 50 mg of clomiphene citrate (CC) therapy whereas 6 women required 100 mg of CC and 3 pregnancies were achieved. This rapid screening method is thus useful in segregating a large number of women according to their ovulatory status and in the subsequent treatment of CLD.

Detection of immunosuppressive activity in human seminal plasma by an immunobioassay.

Recent evidence has shown that under in-vitro conditions human seminal plasma can interfere directly or indirectly with the function of cells of the immune system. It is however, questionable whether the results generated in vitro can be related directly to in-vivo activity. We have therefore standardized an in-vivo immunobioassay to detect the immunosuppressive property of human seminal plasma using adoptive transfer of contact sensitivity to a specific antigen such as dinitrofluorobenzene. Our results indicate that when sensitized lymphoid cells were incubated in vitro with human seminal plasma, their ability to transfer the delayed hypersensitivity in non-sensitized mice was suppressed or inhibited in comparison with the controls. The percentage suppression varied with different samples but the results indicate clearly that the immunosuppressive properties of human seminal plasma can be demonstrated using an in-vivo immunoassay.