Human Oviductal Fluid Research Articles

The new embryo culture medium based on the amino acid concentration of human oviductal fluid enhance the embryo developmental ability; randomized trial

The new embryo culture medium based on the amino acid concentration of human oviductal fluid enhance the embryo developmental ability; randomized trial

The human is an exception to the evolutionarily-conserved phenomenon of pre-fertilization zona pellucida resistance to proteolysis induced by oviductal fluid

Is zona pellucida (ZP) resistance to proteolysis, induced by oviductal fluid (OF), a mechanism common to species other than the pig and cow? ZP resistance to proteolysis induced by OF was observed in the mouse, rat, hamster, rabbit, sheep, goat, pig and cow, but not in humans. Oviductal ZP resistance to proteolysis occurs in the pig and cow where it influences the incidence of fertilization and polyspermy. The effect is observed after incubation of ZP in OFs from pig (pOF), cow (cOF), rabbit (rOF) and sheep (sOF). Oocytes from nine different species, including ungulates, rodents, lagomorphs and primates were incubated in rOF, sOF, gOF, cOF, pOF and human oviductal fluid (hOF). ZP digestion times for the matured oocytes of these nine species, without any treatment or incubated in 5 (mouse, rat, hamster, rabbit, cow, ewe and goat) or 6 (pig and humans) of the OFs collected were compared using three replicates per treatment and at least three oocytes per replicate. In vivo matured oocytes from rat, hamster, mouse, rabbit and humans, in vitro matured oocytes from cow, goat, ewe and pig and rOF, cOF, gOF, sOF, pOF and human (hOF) were collected and processed for the study. Oocytes from each species were incubated in the different OFs for 30 min. The resistance of the ZP of the oocytes to enzymatic digestion in a pronase solution (0.5% in PBS) was measured and registered as ZP digestion time. rOF increased ZP resistance to proteolytic digestion in the range of between 96 and 720 h for any of the species tested, whereas the corresponding increase in human ZP was only 1 min. OFs from the remaining species also had a significant effect, with variations among the cross-species experiments (P < 0.05). hOF, which was only tested on human and porcine oocytes, had no effect on ZP chemical hardening. Measurements of ZP digestion times are not of extreme accuracy and errors of a few seconds can be assumed in the experimental data. However, when differences are in the range of hours among treatments, variations measured in seconds do not alter the robustness of the findings. Human oocytes and OF were of limited access, compared with oocytes from species collected in slaughterhouses. OFs from mouse, rat and hamster were not tested due to the small size of the genital tract in these species and the small volume of fluid available. Since oviductal modification of ZP resistance to proteolytic digestion has been demonstrated to influence fertilization and this pre-fertilization mechanism is considered to contribute to the control of polyspermy, the apparent absence of this mechanism in humans suggests that the regulation of polyspermy depends mainly on other mechanisms, most probably of cortical granule origin. Investigation into a possible relationship between the lack of oviductal ZP hardening in human oocytes and the existence of tubal ectopic pregnancies in this species is proposed. This work was supported by the Spanish Ministry of Science and Innovation and FEDER, Grant AGL2009-12512-C02-01-02. The authors declare no competing interest.

Hepatocyte growth factor in human milk and reproductive tract fluids.

Despite evidence indicating a role for hepatocyte growth factor (HGF) in gastrointestinal and reproductive physiology, the concentration and distribution of HGF in human breast milk (BM) and reproductive tract fluids remain unknown. Using enzyme-linked immunosorbent assay (ELISA), the HGF concentrations were determined in human oviductal fluid (hOF), follicular fluid (FF), amniotic fluid (AF), seminal plasma (SP), and colostrum/milk samples, and expression of HGF mRNA by milk cells and AF cells were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). HGF is present at nearly 70-fold normal serum (0.85+/-0.15 ng/mL) concentration in FF (n = 3; x = 57+/-16 ng/mL) and AF (n = 17; x = 57+/-26 ng/mL), and is also present in hOF (n = 3; x = 4.8+/-2.3 ng/mL) and CVL (n = 8; x = 0.7+/-1.1 ng/mL) varying throughout the menstrual cycle. HGF is found at 3-times serum concentration in BM (n = 24; x = 2.3+/-1.3 ng/mL) with no significant difference between premature and full term or stage of lactation (colostrum, transitional, mature milk). HGF mRNA was detected in BM cells but not in AF cells. HGF is present in sufficient amounts to profoundly affect gastrointestinal maturation in the fetus via swallowed AF and neonate via BM, and helps to explain the increased rate of necrotizing enterocolitis (NEC) in infants of premature rupture of membrane (PROM)-complicated pregnancies, and the decreased rate in breast fed neonates. HGF in FF may be necessary for the development and maturation of the oocyte. HGF in hOF, SP, and cervicovaginal lavage (CVL) is likely to enhances epithelial cell integrity and the mucosal barrier. Thus, HGF is widely available in the reproductive tract with functions that remain to be fully elucidated.

Binding of a bovine oviductal fluid catalase to mammalian spermatozoa.

Hydrogen peroxide (H2O2) is a reactive oxygen species that at low concentration is toxic to sperm. H2O2 inhibits not only sperm viability but also the acrosome reaction, sperm-egg binding, and oocyte penetration. Catalase activates the decomposition of H2O2 into water and oxygen, thus removing an initiator of free radical chain reactions leading to lipid peroxidation. Since the oviduct is known to enhance sperm survival, we hypothesized that it might secrete catalase. We found that oviductal fluid, harvested from washed cells collected at the slaughterhouse, possessed catalase-specific activity that varied during the estrous cycle. Catalase activity increased during the cycle and reached its maximal level just before ovulation (Days 18-20). No significant difference in activity was seen between fluid from the isthmus and that from the ampulla. Indirect immuno-staining of spermatozoa incubated in the oviductal fluid revealed the association of catalase in the region of the acrosomal cap. Addition of a commercial antibody directed against bovine liver catalase completely inhibited catalase activities from the oviductal fluid. Catalase activity was also detected in porcine oviductal fluid, human oviductal fluid, and cervical mucus. Western blots of oviductal fluid probed with the anti-catalase antibody revealed two major bands at 60 and 40 kDa. An immunoaffinity column was used to purify oviductal catalase, showing a unique band at about 60 kDa when analyzed by SDS-PAGE. The purified protein was incubated with bovine, boar, and human sperm, and Western blots of these sperm after several washes detected a band at 60 kDa, indicating that the protein was bound to sperm membranes. However, bovine liver catalase did not bind to sperm. Since H2O2 is one of the key reactants in the chain reaction of free radical production, this enzyme may play an important role in sperm survival within the female tract.

Cytokines of the human reproductive tract.

How is it possible that the female genital tract immunologically does not reject spermatooa not the preimplantation and nidating embryo? Four fluids of the human reproductive tract, i.e., human oviductal fluid (hOF), follicular fluid (FF), amniotic fluid (AF), and seminal plasma (SP) were investigated by specific ELISA for 18 cytokines. The concentrations, presence or absence of these compounds were evaluated for their possible role in the immunology of the reproductive process. Stem cell factor and IL-11 were detected in all reproductive tract fluids examined whereas large amounts of IL-1 beta and IL-1RA was found in AF and hOF. Follicular fluid revealed IL-2. HOF contained IL 2, IL-6, IL-8, TNF-alpha, MIP-1 alpha, IFN-gamma, and high levels of IL-1 beta, IL-10, IL-1RA, and sIL-2R. Amniotic fluid contained sIL-2R, IL-8, IL-1 beta, IL-1RA, IL-6, TNF-alpha, and MIP-1 alpha. No IL-12 or IL-13 was detected in hOF follicular fluid or amniotic fluid. Almost no free TGF-beta 1 or TGF-beta 2 was found in any reproductive tract fluid except seminal plasma. Seminal plasma contained large quantities of free TGF-beta 1 (9,220 +/- 3,635 pg/mL) in addition to large quantities of latent TGF-beta 2 (2,933 +/- 2,169 pg/mL) and TGF-beta 1 (71,000 +/- 3,240 pg/mL). Furthermore, considerable concentrations of IL-8 (1900 +/- 374 pg/mL) and sIL-2R (350 mu/mL) exist in seminal plasma. HOF contains a high level of IL-10 (588 +/- 304 pg/mL), a powerful immune suppressor which probably plays a role in regulating immune responses in the fallopian tube and possibly in the endometrial cavity. Our observations suggest that seminal plasma with its huge content of TGF beta provides immune protection for sperm. Unfortunately, such high concentrations of TGF beta may also inhibit an immune defense in any organ in which semen is deposited.

The sequential effects of human cervical mucus, oviductal fluid, and follicular fluid on sperm function

To compare the sequential effects of human cervical mucus (CM), oviductal fluid, and follicular fluid (FF) on sperm motility, hyperactivation, and the acrosome reaction during 9 hours of incubation in vitro. Spermatozoa from a fertile donor were allowed to penetrate human CM in vitro and then were recovered and incubated in either 30% human oviductal fluid or a simple salt solution for 3 hours before challenge with 20% FF. Thus, the sequential effects of the fluids collected from the female reproductive tract on sperm hyperactivation and acrosome reaction were examined. The donor insemination program at the University Clinic within the Jessop Hospital for Women, Sheffield, United Kingdom. All donors used in this study were involved in the donor insemination program. Sperm motility, hyperactivation, and percentage of acrosome-reacted spermatozoa were measured. This study used an in vitro technique to mimic sperm transport in vivo. Spermatozoa sequentially incubated with female reproductive tract secretions in vitro showed a maintenance in the level of FF-induced hyperactivated motility and a decreased response to the FF-induced acrosome reaction when compared with sperm challenged with FF in the absence of prior incubation with human oviductal fluid. It is suggested that human oviductal fluid may serve to delay the FF-induced acrosome reaction.

Human oviductal fluid prolongs sperm survival

To compare the effect of human oviductal fluid on sperm motility and hyperactivation during 9 hours' incubation in vitro with follicular fluid (FF) and medium controls.Fertile donor spermatozoa were allowed to penetrate human cervical mucus in vitro and then recovered and incubated in either 30% human oviductal fluid, 20% FF, or medium for up to 9 hours. Sperm motion characteristics were measured using a sperm motility analyzer.The donor insemination program at the University Clinic within the Jessop Hospital for Women, Sheffield, United Kingdom.All donors used in this study were involved in the donor insemination program.Sperm motility, hyperactivation, curvilinear velocity, progressive, lateral head displacement, and linearity were measured using a sperm motility analyzer.After 9 hours' incubation, spermatozoa in human oviductal fluid had a significantly higher percentage motility than sperm incubated in FF or the control medium. A more linear sperm motion was consistently observed in the spermatozoa incubated in human oviductal fluid: significantly different from FF and media at 3 hours and 6 hours. The effect of human oviductal fluid on maintaining sperm motility was not affected by the addition of P.Human oviductal fluid can maintain sperm motility in a mechanism that is not mediated by the low concentration of P. We suggest that human oviductal fluid is a favorable environment for sperm survival.

Human oviductal fluid proteins. V. Identification of human oviductin-I as alpha-fetoprotein

To investigate whether human oviductin-I (hOV-I) from human oviductal fluid (hOF) and human alpha-fetoprotein (hAFP) are identical molecules. Comparison of amino acid and carbohydrate composition of hOV-I with that of hAFP. Isolation of hAFP from oviductal fluid of a patient and evaluation of its immunochemical properties in relation to hOV-I and authentic hAFP standard. Procedures were performed in an academic research environment. Human oviductin-I was isolated from a pooled sample of hOF obtained from four patients in a third world country. AFP was purified from a sample of oviductal fluid obtained from a single patient during her periovulatory period. Human oviductal fluid was collected coincident to elective tubal ligations. The amino acid and carbohydrate composition of hOV-I was determined. AFP was purified from hOF using ion-exchange chromatography and gel filtration. The identity between hOV-I and hAFP from hOF was assessed using enzyme-linked immunosorbent assay (ELISA) and Western immunoblot analysis. The amino acid and carbohydrate composition of hOV-I indicated similarities between hOV-I and hAFP. Both hOV-I and hAFP reacted with mouse anti-hAFP monoclonal antibody and purified polyclonal rabbit anti-hOV-I immunoglobulin G in an identical manner as observed by noncompetitive ELISA. Purified AFP from hOF of a single patient had a molecular mass of 66 kd and was found to be identical with hOV-I by Western immunoblot analysis. Human oviductin-I and hAFP are chemically similar and immunologically identical molecules. We believe that this finding is the first documentation for the existence of AFP in hOF.

Human oviductal fluid proteins. VI. Correlation between alpha-fetoprotein and serum levels of ovarian hormones

To determine the relationship, if any, between the ovarian hormonal status of women and levels of alpha-fetoprotein (AFP) in their oviductal fluid. The threshold questions were the following: (1) does the presence of AFP in human oviductal fluid (hOF) represent a generalized phenomenon and (2) if so, is there any relationship between AFP in hOF and the ovarian hormones, estradiol (E2) and progesterone (P)? Eleven women who elected minilaparotomy tubal ligations volunteered to donate hOF and serum samples for this study. AFP, E2, and P were determined be radioimmunoassay. Procedures were performed in an academic research environment. This study encompasses only those patients who elected minilaparotomy tubal ligation and volunteered to donate hOF and sera. Clinical features of these patients are described. Patients undergoing tubal ligations had Foley catheters inserted into the fimbriated ends of each oviduct, and hOF was collected for 24 hours. Concomitantly, blood was drawn for analyses of serum. The Investigational Review Board of the Erie County Medical Center approved this study requiring the collection of oviductal fluid from human subjects. Levels of AFP in hOF did not correlate with the serum concentrations of E2 and P per se. However, there was a highly significant correlation between levels of AFP in hOF and serum E2:P ratio. The concentration of AFP in hOF progressively increased with respect to the ratio of serum E2:P. Although AFP was present in all samples of hOF, it was undetectable in corresponding sera. Based on these data, it is concluded that AFP is generally present in hOF. Furthermore, it is suggested that AFP in hOF may be under the control of ovarian steroids.

Human oviductal fluid (hOF) proteins. IV. Evidence for hOF proteins binding to human sperm

In the management of infertile patients, it is well recognized that gamete intrafallopian transfer (GIFT) has a higher success rate than in vitro fertilization and embryo transfer. Oviducts produce unique proteins that may be responsible for the success of GIFT. Unique proteins from human oviductal fluid (hOF) have been purified in the authors' laboratory. One of these proteins of 54 kDa molecular weight, and containing carbohydrate, was obtained in a highly purified state. In this study, human sperm were incubated with: (1) a mixture of hOF-specific proteins, and (2) the highly purified hOF glycoprotein, designated human oviductin I (hOV-I). Using indirect immunofluorescence, the authors studied the ability of hOF proteins to bind to human sperm. The mixture of hOF proteins appeared to bind to the surface of the entire sperm. Conversely, hOV-I binding was restricted to the head region only. Studies are in progress to discern the function of the sperm-binding interaction(s) with hOF proteins.

Human oviductal fluid proteins. III. Identification and partial purification

The role of the macromolecular constituents of human oviductal fluid (hOF) in reproductive physiology is poorly understood. Oviductal fluid contains proteins derived by transudation from serum and those synthesized and secreted by the tubal mucosa. In this communication, we describe purification of hOF-specific proteins. As a first step, serum proteins were removed with an immunoaffinity gel. Fractionation of the proteins unique to hOF was accomplished with DEAE-cellulose chromatography. One of these proteins was obtained in a highly purified state after gel filtration on G-75 Sephadex. This protein of 54 kDa molecular weight had a pI of 4.5 and contained carbohydrate. We propose that this hOF glycoprotein be designated "human oviductin I" (hOV I).

Human oviductal fluid proteins. II. Preparation of an antiserum to a human oviductal fluid protein: existence of autoantibodies against it in some sera

Rabbit antisera elicited by injection of human oviductal fluid (HOF) and rendered specific by absorption with human kidney and human serum revealed the presence of two possibly HOF-specific antigen(s). One antigen was detected by immunodiffusion in 14 of the 14 fluids tested with one of these antisera. HOF-specific antigen was shown to be a beta-globulin by immunoelectrophoresis. A second antigen was seen in two samples. That autoantibodies might exist directed against HOF-specific antigen in the donor's serum was suggested by immunodiffusion experiments in which 4 of the 13 sera tested gave lines of precipitation when reacted against the corresponding oviductal fluid. In one instance, this line was the same as one of the two lines seen in tests using rabbit antiserum.

Human oviductal fluid proteins

The popularity of mini-laparotomy provided an opportunity to easily collect human oviductal fluid (HOF). Volumes of HOF produced by two oviducts per 24 hours correlated positively with serum estradiol determinations, while protein concentration in HOF was inversely proportional to estrogen levels. Estrogen appeared to stimulate the production of oviductal fluid in women. The greatest volumes of HOF were observed at midcycle, coincident with the estrogen peak. Protein concentration was lowest at the time of ovulation and highest immediately before and after menstruation. In a large percentage of patients, certain proteins made their appearance at the time of ovulation and receded or disappeared within three to five days thereafter. By electrophoresis, these proteins were seen in the albumin region and in the beta globulin region.

Prostaglandins in the Human Fallopian Tube

The concentration of prostaglandin (PG) F2 alpha in human oviductal fluid was determined, and the histological localization of PGF2 alpha was correlated with the menstrual cycle to illuminate possible roles of oviductal PGF2 alpha in human luteolysis. Human oviductal fluid was collected preovulatorily, ovulatorily, and postovulatorily. PGF2 alpha was radioimmunoassayed and it was found that the levels in oviductal fluid were higher (5-7.1 ng/ml) than those normally found circulating in serum (5 ng/ml, but these authors did not test sera for PGF2 alpha levels). Using immunofluorescence of biopsy material from patients undergoing elective tubal ligations, PGF2 alpha could be histologically localized in the human oviductal mucosal surface before ovulation; after ovulation, the PG was found in oviductal lamina propria, but no PHF2 alpha was found in any portion of a postpartum or postmenopausal oviduct in this study.