Comparative concentrations of steroid hormones and proteins in human peri-ovulatory peritoneal and follicular fluids

Despite the fact that both peritoneal (PF) and follicular (FF) fluids have a common ovarian origin, FF is a natural inducer of sperm acrosome reaction (AR) while PF is not. To better understand these effects, concentrations of oestradiol, progesterone and proteins in peri-ovulatory PF and FF were determined and compared. PF was aspirated by laparoscopy at the peri-ovulatory stage from women with unexplained infertility. FF was collected from patients undergoing IVF and pooled. PF and FF were tested for the presence of antisperm antibodies. Oestradiol and progesterone were measured by enzyme immunoassay, and total protein concentration was determined and analysed. The AR was determined in spermatozoa that were exposed to PF alone, progesterone-supplemented PF, progesterone, control medium, or ethanol. No antisperm antibodies were found in any fluid tested. Oestradiol and progesterone and concentrations in PF were significantly lower than in FF. Protein concentration was also significantly lower in PF than in FF, but no differences were observed between the electrophoretic patterns. When capacitated spermatozoa were exposed to progesterone-supplemented PF there was a significant increase in the percentage of AR with respect to those in PF, control medium or ethanol. These results suggest that the lack of AR-stimulating activity of PF was related to its lower progesterone concentration compared with FF.     

ELISA for antisperm antibodies in unexplained infertile women

It is commonly believed that autoimmune or isoimmune responses to human sperm antigens are associated with human infertility. We examined an enzyme linked immunosorbent assay (ELISA) kit used in the detection of antisperm antibodies in the sera of women with unexplained infertility. 1) For the ELISA assay, an absorbance at 405nm greater than 0.300 was considered positive. Of the 83 sera of infertile women studied, 20(24.1%) were positive. This value was significantly higher than that of the control group: 10% of pregnant women, 11.1% of unmarried women and 10% of healthy men (p less than 0.05). 2) The results obtained with ELISA were compared with those of the sperm immobilization test. Comparison of the results of ELISA with those of the sperm immobilization test indicated that these methods detect a different, though often overlapping, spectrum of antibody activity. 3) The results obtained with ELISA did not always correlate with those of the Huhner test. However the ELISA method may be useful in the clinical screening of antisperm antibodies because of its simplicity and rapidity.     

Certain immunological relationships between the pancreatic kallikrein and spermatozoa

The immunological relationship between kallikrein and acrosomal extracts from bull, ram, boar, and human spermatozoa was studied. Highly purified porcine pancreatic kallikrein was used and rabbit antikallikrein serum was produced. The immunochemical analysis did not reveal any antigenic relationship between kallikrein and the acrosomal extracts from spermatozoa of these species. Preincubation of spermatozoa in rabbit antikallikrein serum did not induce any changes in the acrosomal proteolytic activity. The antikallikrein serum significantly inhibited the motility of bull and boar spermatozoa but did not exert any marked influence on the motility of ram and human spermatozoa.     

Albumin and the acrosome change in mammalian spermatozoa

Samples of follicular fluid were collected from ovaries of women, rabbits, cats, pigs, and cows, and examined for their effects on the acrosome of rabbits' spermatozoa. The occurrence of the acrosome reaction was assessed by staining. All follicular fluids were effective in inducing the reaction. Those taken from humans, cow, or pig follicles were more effective than those from cats or rabbits. The factor in follicular fluid was a protein of large molecular weight, and its properties were dependent on the presence of calcium ions. The reactive protein in bovine follicular fluid evoked the acrosome reaction in hamster spermatozoa. The protein may have a role in vivo although other studies in vitro have indicated that it can be replaced by the use of other compounds in culture media used for capacitation and fertilization.     

Ovarian Dysfunction in Endometriosis-Associated and Unexplained Infertility

Purpose: The impact of endometriosis and unexplained infertility on follicular function and fertilization of oocytes in cycles totally unperturbed by exogenous gonadotrophins, when compared with controls with tubal damage, were examined. Methods: In natural cycles, without any exogenous gonadotropins, endocrine and ultrasonographic studies of follicular maturation in 18 women with minor endometriosis (41 cycles), 15 women with unexplained infertility (31 cycles), and 34 women with tubal damage (88 cycles) were performed. Results: The endometriosis group had a significantly longer follicular phase (median: 15,13, and 13 days). Both endometriosis and unexplained infertility had significantly reduced LH concentrations in follicular fluid compared with tubal damage (median: 12.1, 11.5, and 15.9 IU/L, respectively). Endometriosis was associated with a significantly reduced fertilization rate compared with unexplained infertility or tubal damage (46, 65, and 69%, respectively). Conclusions: These data show continuing evidence of ovulatory dysfunction leading to reduced fertilization rates in women with minor endometriosis.     

Effects of follicular fluid and steroid hormones on chemotaxis and motility of human spermatozoa in vitro

The 'Transwell system' was used to test the response of human spermatozoa to human follicular fluid ,progesterone ,estradiol and mifepristone. Motility parameters were assessed with computer-assisted sperm analysis. Follicular fluid and progesterone induced significant accumulation of spermatozoa. Changes compatible with an increased progressive and hyperactivation-like motility were obtained with follicular fluid but not with progesterone. Mifepristone eliminated the progesterone-induced accumulation of spermatozoa but had no significant effect on the accumulation of spermatozoa in wells containing human follicular fluid. Furthermore ,mifepristone abolished the motility changes effected by follicular fluid. Estradiol had no effect on accumulation or motility of spermatozoa. Human follicular fluid exerted a strong effect on sperm chemoattraction and motility in vitro ,while progesterone influenced sperm chemoattraction only.     

Effects of follicular fluid and steroid hormones on chemotaxis and motility of human spermatozoa in vitro.

The 'Transwell system' was used to test the response of human spermatozoa to human follicular fluid, progesterone, estradiol and mifepristone. Motility parameters were assessed with computer-assisted sperm analysis. Follicular fluid and progesterone induced significant accumulation of spermatozoa. Changes compatible with an increased progressive and hyperactivation-like motility were obtained with follicular fluid but not with progesterone. Mifepristone eliminated the progesterone-induced accumulation of spermatozoa but had no significant effect on the accumulation of spermatozoa in wells containing human follicular fluid. Furthermore, mifepristone abolished the motility changes effected by follicular fluid. Estradiol had no effect on accumulation or motility of spermatozoa. Human follicular fluid exerted a strong effect on sperm chemoattraction and motility in vitro, while progesterone influenced sperm chemoattraction only.     

Evaluation of serum-associated embryotoxicity in women with reproductive disorders

Purpose Our purpose was to determine whether some cases of infertility may be due to serological factors inhibiting development of the embryo.Method We examined the effect of infertile women's sera on the expansion, attachment, and spreading of mouse blastocysts in culture. Cell marker expression was also assayed by an indirect immunofluorescence technique. Serum samples from 75 infertile women were compared to the effect of 24 control AB sera.Results After 72 hr, blastocyst spreading was significantly different depending on whether cultured in sera from women with unexplained infertility, anovulatory infertility, diethylstilbesterol exposure or controls. Neither sera from women with mechanical infertility (14) nor sera from women with endometriosis (8) affected blastocyst growth in culture.Conclusions Inhibitory sera were capable of reducing cytokeratin expression but had no effect on placental alkaline phosphatase or concanavalin A expression by blastocyst cells. It can be inferred that the inhibitory effect of sera from women with certain types of infertility might be due to damage to the cytoskeleton. This in vitro assay may predict the success or failure of IVF.     

Peritoneal fluid from patients with unexplained infertility inhibits growth of 2-cell mouse embryos.

A significantly higher DHEAS concentration was measured in the peritoneal fluid of unexplained infertility patients (1171.4 +/- 155 ng/mL) in comparison to normal controls (667.6 +/- 82 ng/mL). Since the androgenic male serum does not promote blastocyst formation in the mouse embryo assay system, the potential of growth impairment by peritoneal fluid (PF) obtained from 22 women with unexplained infertility and 10 fertile controls was assessed. Where peritoneal fluid and serum from unexplained infertile (UI) patients were used as media supplement in mouse embryo culture, a significant inhibition of growth was observed in dishes containing PF but not serum. When DHEAS was added in varying concentrations to the culture media, a dose-dependent inhibition of embryo growth was observed. These findings show that the elevated DHEAS concentrations in the PF of UI patients adversely effect embryo growth and further suggest that increased DHEAS levels in the cul-de-sac fluid may be a causative factor for infertility.     

A simple method of isolation of human spermatozoa with the swim-up technique: usefulness of various chemically defined media,and influence of porcine follicular fluid

The aim of the study was to define minimal conditions necessary to isolate human spermatozoa using their swim-up potential. The efficiency of the isolation of spermatozoa from seminal fluid was tested after 1 hour incubation at 37 degrees C of seminal plasma together with isolation mediums, at the volume proportion 2:1. Three different media were tested: RPMI 1640, earle solution, and 0.9% solution NaCl. In the other experiments seminal fluid or RPMI 1640 were enriched with porcine follicular fluid and were incubated together. Sperm density and percentage of progressively motile sperms as well as their viability did not differ depending on the medium used. However, migration was weaker at higher sperm density. Addition of porcine follicular fluid to both seminal fluid and isolation medium lowered the efficiency of sperm migration to the isolation medium.     

Modulation of human sperm function by peritoneal fluid

OBJECTIVE: To examine the effect of peritoneal fluid on various parameters of sperm function in vitro. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Semen samples were obtained from normozoospermic volunteers (n = 43). Peritoneal fluids were aspirated laparoscopically from women with unexplained infertility (n = 14). Follicular fluid and oocytes were collected from patients undergoing IVF-ET. INTERVENTION(S): Sperm incubated under capacitating conditions were exposed to peritoneal fluid, and functional variables were evaluated in vitro. MAIN OUTCOME MEASURE(S): Sperm viability and motility, follicular fluid and calcium ionophore-induced acrosome reactions, protein tyrosine phosphorylation, expression of D-mannose binding sites, and ability of sperm to interact with zona pellucida. RESULT(S): Exposure of sperm to peritoneal fluid for up to 6 hours did not affect sperm viability or motility. Unlike follicular fluid, peritoneal fluid did not induce the acrosome reaction. Moreover, incubation of sperm with > or =20% v/v peritoneal fluid for 1 hour prevented the follicular fluid and the ionophore-induced acrosome reaction. Although treatment with peritoneal fluid allowed protein tyrosine phosphorylation during capacitation, it resulted in a significant decrease in the expression of D-mannose binding sites and sperm-zona pellucida binding. CONCLUSION(S): Peritoneal fluid maintains sperm survival and decreases sperm ability to respond to inducers of the acrosome reaction and bind to the zona pellucida in vitro, indicating that this fluid might modulate sperm function in vivo.     

Blocking of human fertilization in vitro by sera with sperm-immobilizing antibodies

Serum samples with sperm-immobilizing antibody activity from six women were examined for ability to block sperm-egg interaction by a zona penetration test where human follicular ova matured in vitro were used. Exposure of spermatozoa from a fertile healthy donor to the sera impaired binding to and penetration through the zona pellucida of the spermatozoa completely in five cases and incompletely in one case. Successful fertilization in vitro was achieved by using fetal cord serum instead of autoserum of the patient included in the in vitro fertilization and embryo transfer program. These results suggest that interference with sperm-egg interaction may be an additional mechanism of infertility that is caused by antisperm antibodies.     

The detection in human sera of antisperm antibodies reactive with FA-1, an evolutionarily conserved antigen, and with murine spermatozoa

Evolutionarily conserved antigens are present on spermatozoa of several mammalian species. We tested sera from infertile men and women containing antisperm antibodies (ASAs) for their reactivity with FA-1, an antigen known to be present on murine and human spermatozoa. Fifty percent of male sera and 63% of female sera contained anti-FA-1 antibodies, as judged by enzyme linked immunosorbent assay (ELISA). Fourteen percent of male sera and 50% of female sera were also shown to possess ASAs reactive with living mouse spermatozoa, and murine in vitro fertilization was inhibited by human antibodies. These results suggest that the transfer of immunoglobulins from human sera to spermatozoa of other species may provide a model to study how ASAs effect sperm function.     

Occult ovulatory dysfunction in women with minimal endometriosis or unexplained infertility

Characteristics of follicular development and hormonal patterns were evaluated in 17 women with minimal endometriosis and 11 with unexplained infertility. The controls were 7 women with male factor infertility and 8 who conceived during an investigational cycle. Women with minimal endometriosis had more and smaller follicles at luteinizing hormone (LH) surge, lower preovulatory estradiol (E2), and lower E2 at LH surge. Women with unexplained infertility had lower LH surges and a trend to a shorter follicular phase. Occult ovulatory dysfunction and may contribute to infertility in women with minimal endometriosis or unexplained infertility.     

Ovarian follicular fluid contains immunoreactive estriol: lack of correlation with estradiol concentrations.

Estradiol and estrone concentrations in ovarian follicular fluid change according to the ovulatory cycle, but no studies on the possible presence and/or changes of estriol are available. The aim of the present study was to evaluate whether estriol is measurable in follicular fluid and how its concentration changes according to the volume of ovarian follicles and to the maturational stage of oocytes. A group of women (n = 39) undergoing a program of induction of ovulation was included in this study and divided into three groups according to the causes of infertility: those with unexplained infertility (n = 11); those with endocrine disturbances (n = 5); and normal ovulatory women (n = 23) (controls). The follicles recruited (n = 116) on the basis of morphology and the appearance of the oocyte cumulus-corona complex were divided into: mature (n = 22); intermediate (n = 75); immature (n = 11); and atretic (n = 8). Ovarian follicles were also divided according to the diameter of each: < 1.5 cm (n = 38); 1.6-2.4 cm (n = 66); and > 2.5 cm (n = 12). Ovarian follicular fluids were aspirated under ultrasound guidance and a blood specimen was collected from each subject. Estriol and estradiol concentrations were evaluated by radioimmunoassay in serum and follicular fluid following an ether extraction. Estriol was found in high concentration in each sample of follicular fluid, significantly higher than in the respective serum sample (p < 0.01). Although the estradiol concentration was significantly lower in follicles containing immature and atretic oocytes than in intermediate or mature follicles (p < 0.01), the estriol concentration did not depend upon the maturational stage. In addition, the follicular fluid estriol concentration did not differ according to the causes of infertility. Follicular fluid and serum estradiol concentrations showed significant correlation (p < 0.01), whereas no significant correlation was observed between serum and follicular estriol concentrations. The present data show that follicular fluid contains a high concentration of estriol and that its changes are independent of the ovulatory cycle and estradiol concentrations, supporting an independent origin and suggesting a different function for estriol.     

Do alterations in follicular fluid proteases contribute to human infertility?

Purpose

Cathepsin L and ADAMTS-1 are known to play critical roles in follicular rupture, ovulation, and fertility in mice. Similar studies in humans are limited; however, both are known to increase during the periovulatory period. No studies have examined either protease in the follicular fluid of women with unexplained infertility or infertility related to advanced maternal age (AMA). We sought to determine if alterations in cathepsin L and/or ADAMTS-1 existed in these infertile populations.

Methods

Patients undergoing in vitro fertilization (IVF) for unexplained infertility or AMA-related infertility were prospectively recruited for the study; patients with tubal or male factor infertility were recruited as controls. Follicular fluid was collected to determine gene expression (via quantitative polymerase chain reaction), enzyme concentrations (via enzyme-linked immunosorbent assays), and enzymatic activities (via fluorogenic enzyme cleavage assay or Western blot analysis) of cathepsin L and ADAMTS-1.

Results

The analysis included a total of 42 patients (14 per group). We found no statistically significant difference in gene expression, enzyme concentration, or enzymatic activity of cathepsin L or ADAMTS-1 in unexplained infertility or AMA-related infertility as compared to controls. We also found no statistically significant difference in expression or concentration with advancing age.

Conclusions

Cathepsin L and ADAMTS-1 are not altered in women with unexplained infertility or AMA-related infertility undergoing IVF, and they do not decline with advancing age. It is possible that differences exist in natural cycles, contributing to infertility; however, our findings do not support a role for protease alterations as a common cause of infertility.     

Mixed lymphocyte culture responses in infertile couples.

Twenty couples suffering from unexplained infertility have been tested in mixed leucocyte culture to determine if the wives were preimmunized against their husbands. The wives were tested against their husbands and four unrelated persons, and thymidine incorporation was measured on the third and fifth days of culture. No evidence was found that would indicate a preimmunization of the wife against her husband as an explanation for their infertility. A slight MLC stimulatory capacity was found in sera from infertile women.     

VEGF, TNF-alpha and IL-6 in the peritoneal fluid of women with unexplained infertility

Unexplained infertility is an important problem in diagnosis and therapy in everyday gynecologist's practice. Looking for possible reasons of the unexplained infertility we studied the concentrations of selected cytokines (VEGF, TNF-alpha and IL-6) in the peritoneal fluid of women suffering from the unexplained infertility. We compared the results in the studied group with the control group and with the patients with endometriosis. Immunological disorders of the peritoneal fluid in endometriosis are thought to take part in its pathomechanism. Our results suggest that the levels of one of the main factors of endothelium proliferation (VEGF) in the peritoneal fluid from women with unexplained infertility and women with endometriosis are comparable. Concentration IL-6 and TNF-alpha in the peritoneal fluid in case of unexplained infertility and control group was lower than in the endometriosis patients.     

Sperm function in patients with unexplained infertility

Summary. Sperm function was studied in 27 patients with hitherto unexplained infertility. The ability of spermatozoa to reach the site of fertilization was assessed by laparoscopic sperm recovery from the peritoneal fluid and fimbrial rinsings and sperm fertilizing capacity with the zona-free hamster egg penetration in vitro test. The ability of spermatozoa to reach the site of fertilization correlated significantly with their fertilizing capacity in vitro , but was totally unrelated to any of the conventional criteria of semen quality, including the postcapacitation movement characteristics of the spermatozoa. Among patients with unexplained infertility, there are individuals with defects of sperm function which cannot be identified by conventional clinical techniques.     

Lymphocyte activity in the presence of peritoneal fluid from fertile women and infertile women with and without endometriosis

Peritoneal fluid from women with endometriosis, unexplained infertility, and fertile controls were compared to one another and to normal human serum for effects on lymphocyte proliferation in vitro. Peritoneal fluid samples were also assayed for both interleukin-1 and interleukin-2. All peritoneal fluid samples significantly enhanced lymphocyte proliferation in both mitogen-stimulated and unstimulated cultures compared with serum controls. Mitogen-induced leukocyte proliferation was higher in the presence of peritoneal fluid from women with endometriosis compared with other samples. Five out of 23 samples from endometriosis patients contained elevated levels of interleukin-1 and three out of 23 contained elevated levels of interleukin-2. Six out of eight peritoneal fluid samples from unexplained infertility patients also had elevated levels of interleukin-2; samples from fertile women did not contain elevated levels of either cytokine. Our data indicate that peritoneal fluid from women with endometriosis and unexplained infertility support the activation and proliferation of lymphocytes. Leukocyte products may locally affect the progression of disease and fertility.