Diaa M. El-Mowafi - Zagagig University, Egypt

Peritoneal fluid mediated embryotoxicity in unexplained infertility

Diaa El-Mowafi Associate Professor*, Umnia El-Hendy Assistant Professor, Roshdan Arafa Professor, Wafaa El-Mosallamy Associate Professor, Emad Basiony Assistant Professor, and El-Sayed Abdel Ghany Assistant Professor
Obstetrics & Gynaecology* and Microbiology &Immunology Departments, Benha Faculty of Medicine, Egypt

Abstract

Objective To study the effect of peritoneal fluid (PF) on in vitro early embryogenesis.

Design Prospective study.

Patients PF samples were collected through laparoscopy from 60 women. Thirty women had unexplained infertility and other 30 fertile women were in need of laparoscopic tubal sterilization and considered as a control group.

Methods The PF samples were subjected to (1) estimation of total protein concentration, (2) detection of different protein bands by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), (3) qualitative estimation of TNF-a by Western blotting, and (4) study its effect on in vitro cleavage of 2-cell mouse embryos.

Results: The total protein concentration was significantly higher in PF of unexplained infertility group (mean 27.4 mg/ml) than in PF of control group (mean 21.07 mg/ml) (P < 0.05). Nearly, the same protein bands were detected by SDS-PAGE in PF of both groups. A peritoneal fluid band of 32 kd appeared in 5/30 cases of unexplained infertility and in non of the control group. TNF-a band was detected by western blotting in all cases of unexplained infertility and in 7/30 cases of fertile control group. The embryotoxicity of PF of unexplained infertility was significantly high (P<0.05). Embryos reached to the blastocyst stage in 2-cell mouse embryos exposed to PF of unexplained infertility and control groups were 220/310 and 280/300 respectively.

Conclusions: PF from women with unexplained infertility contained high protein concentration and a band of cytokine (TNF-a ). Also it was more toxic to 2-cell mouse embryos than peritoneal fluid from fertile women.

Introduction

Infertility is a serious complaint affecting about 15% of married couples and has its social and psychic impacts. Unexplained infertility is a state in which no cause could be detected to explain it in spite of thorough investigations for both partners. Unexplained infertility remains a frustrating condition for both patient and clinician alike1,2.

One of the possible causes of «unexplained infertility» may be an immunological problem, related to antisperm antibodies, humoral, or cell mediated immune disorders3. It may be hidden in the peritoneal fluid, away from routine sites of investigations. The attention was directed to the peritoneal fluid microenvironment that is surrounding the ovary and proximal part of the tubes and passes easily into the tubal lumen, the portion in which the normal fertilization takes place. Spermatozoa as well as early embryonic stages are exposed to the peritoneal fluid with its cellular and soluble components4. Macrophages appear to play a leading role in infertility with contributions from other cell types such as peritoneal mesothelium, cell lymphocytes and even endometrium itself as in cases of endometriosis. Accumulated soluble substances such as prostaglandin, cytokines and growth factors probably act as mediators to induce endometriotic support and interference with infertility and early embryonic development5.

The peritoneal mediators of infertility include macrophages6, Tumor necrosing factor7markers of inflammatory response and prostaglandins8,9.

The purpose of this study was to evaluate the effect of PF from patients with unexplained infertility on in vitro cleavage of 2-cell mouse embryos and to analyze the protein content of PF in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to determine if the protein content and the cytokine (TNF-a ) have a relation to unexplained infertility.

Patients, Materials and Methods

Patients: This study was carried out on 60 patients attending the gynecology departments in Benha University Hospitals, Egypt and Hutzel Hospital, Wayne State University, Detroit, Michigan, USA. The patients were divided into 2 groups, each consists of 30 patients, group I (GI) patients with unexplained infertility and group II (GII) fertile patients indicated for laparoscopic sterilization as a control group. Thorough infertility investigations were done to GI to exclude any cause of infertility including complete semen analysis, post-coital test, premenstrual endometrial biopsy, hysterosalpingography and diagnostic laparoscopy. The fertile control group included non-smoking, non-lactating, parous women. They were neither taking oral contraceptive nor using intrauterine contraceptive device in the last 3 months and were candidates for elective laparoscopic tubal sterilization.

A complete history was taken from each patient including history of exposure to infection, previous medications or surgical interference. All women were subjected to general, abdominal and local examination.

Samples: Peritoneal fluid (PF) samples were collected by the gynecologic surgeon from the anterior and posterior cul-de sacs by an aspiration needle during laparoscopy before any manipulative procedures or tubal chromopertubation. Samples were transported in ice to the laboratory within 30 minutes. PF samples straw in color were centrifugated (1500 rpm) for 10 minutes at 4° C. The supernatant free cells were stored in aliquots of 25m l volume at -70° C till the time of assay for: estimation of total protein concentration, analysis by sodium dodecyl sulphate (SDS) ployacrylamide gel electrophoresis (PAGE), detection of TNF-a and its effect on 2-cell mouse embryos development.

Materials:

Animal: B6 CBAF1/J female and male mice (Jackson, Lab, Ontario, Canada).

Reagents, Solutions and kits: Reagents and kits were obtained from the following sources: Ham’s F10, fetal calf serum, light parafin oil, pregnant mare serum, human chorionic gonadotropin (hCG) (Sigma, USA). Bicinchoninic acid protein assay reagent kit (Pierce Chemical Company,USA). 30% acrylamide / 0.8% bisacrylamide, tris cL/SDS pH 6.8, tris cL/SDS pH 8.8, SDS stock (SDS, 20%), ammonium persulphate, TEMED (N,N, N, N tetramethyl enediamine), glycine, glycerol, beta-mercaptoethanol, bromophenol blue, molecular weight marker (97000-17000 kilo dalton), nitrocellulose membrane sheet (0.45 microns), Aurodye, colloidal gold reagent (Bio-Rad- Lab, USA), Polyclonal antihuman TNF-a (Endogen, USA). Goat anti- rabbit IgG-alkaline phosphate conjugate substrate kit (Bio-Rad lab, USA).

Estimation of Protein Concentration in the Pf:

Protein concentration was measured by bicinchoninic acid protein assay reagent kit following the instruction of kit manufacturer and as described by Smith et al., (1985).

The working reagent was prepared by adding 50 parts of reagent A to 1 part of reagent B and 10 ul of each sample was added to 200 ul of working solution in a microtiter plate, incubated for 30 minutes at 37oC and the absorbance was measured at 562 nm on plate reader and the proteins concentration of each sample was measured by standard curve.

Analysis of PF by SDS-PAGE:

Analysis of PF by SDS-PAGE was performed as described previously by Laemmli, (1970). Pouring the separating gel: 10% separating gel (3.3 ml acrylamide stock, 4.2 ml deionized water, 2.5 ml tris cl (pH 8.8), 100 m l SDS stock, 50ul of 10% ammonium persulphate and 10 m l TEMED) was transferred to the center of the glass plate sandwich using a pasteur pipet. The top of the gel was covered with a layer of deionized water, the gel polymerized in about 30 minutes at ambient temperature. The water layer was removed and the staking gel (6.1 ml distilled water, 2.5 ml tris cl pH (6.8), 1.3 ml stock acrylamide, 100 m l SDS stock, 50 m l of 10% amonium persulphate and 10m l TEMED) was poured. A teflon comb was inserted into the layer of the staking gel solution which was allowed to polymerized for 30-45 minutes at room temperature.

Loading the gel; each PF sample was diluted 1:2 (vol/vol) with sample buffer (1ml tris cl/ SDS pH 6.8, 0.8 ml glycerol, 1.2 stock SDS, 0.4 ml b- mercaptoethanol, 0.2 ml 0.05% bromophenol blue solution and completed to 4 ml with H2O) and boiled for 5 minutes at 100° C. The electrophoresis buffer (3gm tris cl, 14.4 gm glycine & 25 ml H2O, pH 8.3) was poured into the lower buffer chamber. The protein samples and molecular weight marker were loaded into the wells. Then the power supply was connected to the cell and run at 45 milliampere of constant current until the bromophenol blue marker reached the bottom of the gel. Carefully, the gel was dissembling from the glass plates where it could be stained with Coomassie brilliant blue solution for 4 hours with agitation. Then the staining solution was removed and the destaining solution was added for 2 hours with agitation and continue until the blue bands and a clear background obtained. The gel was stored in 10% acetic acid and 5% ethanol. To maintain a permanent gel record, the gel must be dried in a conventional gel-dryer at 80° C for 60 minutes.

Western Immunoblotting for Detection of TNF-a (Towbin et al., 1979):

Following electrophoresis, the gel was rinsed in transfer buffer (Tris, 3.03 gm; glycine 14.4 gram and methanol 200 ml completed to 1 liter with distilled water, PH 8.1). The nitrocellulose membrane was cutted to the dimentions of the gel. Saturated filter paper with transfer buffer was placed on the top of the filter pan. The gel was placed on the top of the paper. The surface of the gel was flooded with transfer buffer, the membrane was applied and the sandwich was completed by placing a piece of saturated filter paper on top of the membrane. The power supply was turned to initiate transfer at 100 voltage for 1 hour.

Protein blots on nitrocellulose membrane was stained by Aurodye following the instruction of the kit manufacturer.

At the end of blotting, the membrane was washed extensively with phosphate buffer saline (PBS) for 30 minutes at 37ºC and 3 times for 15 minutes at room temperature. The rinsed membrane was incubated in Aurodye under constant agitation until color formation had reached (Purple color) about 4 hours then the blot was washed with distilled water and dried in air.

Detection of the Cytokine TNF-a on the Nitrocellulose membrane:

TNF-a was detected on the nitrocellulose membrane following the instruction of the manufacturer. At the end of the blotting, the membrane was immersed into the blocking solution, washed, then polyclonal anti-human TNF-a was added to the membrane and incubated overnight. To increase the sensitivity of detection, the membrane was washed, goat anti- rabbit IgG AP conjugate was added to the membrane and incubated for 2 hours with gentle agitation. The membrane was washed twice and immersed in colour development solution for 30 minutes, maximally where purple visible bands appeared.

In Vitro Embryotoxicity Assay of the Peritoneal Fluid (Hogan et al., 1994)

Female mice of B6 CBAFI /J strains were injected intraperitoneally with 5 IU of pregnant mare serum gonadotropin to stimulate follicular growth and 48 hours later with 5 IU of hCG to trigger ovulation. Female mice were giving the chance for matting by keeping them in a cage with male mice for overnight. Forty-eight hours after hCG injection the mice were killed by cervical dislocation and the oviducts were dissected and transferred to a Petri dish containing Ham’s F10 medium supplemented by 10% fetal calf serum, 100 U/ml benzyl penicillin and 100 m g/ml streptomycine sulphate. With one ml syring attached to a 32 gauge needle, the oviduct was flushed to separate the 2-cell mouse embryo. Parafin oil was added to the previous medium which equiliberated at 37oC in 5% CO2 overnight. Thirty m L of the equiliberated medium were dropped on the bottom of the tissue culture dish, at least 10 embryos were transferred in each drop . The tested PF was added in the dilution 1: 1 to culture medium in drops, the dish was floaded with parafin oil and incubated in 5% CO2 in humidified air at 37oC for 72 hours. Embryo developmental stage was evaluated under dissecting microscopy at 400X magnification. The blastocyst formation rate was recorded after 72 hours of in vitro culture. Embryos were classified as degenerated if dark granular cytoplasm, fragments or cell mass retracted from the zona pellucida. The percentage of normal blastocyst to total numbers of embryos in each culture after 24, 48 and 72 hours of development was estimated.

RESULTS

The clinical characters of the 60 patients included in this study as regard age, weight and duration of infertility are demonstrated in Table (1).

Table 1: The clinical characters of unexplained infertility group and fertile control group.

Characters	Unexplained infertility group	Fertile Control group
Age / year mean ± SD range Weight (kg) mean ± SD range Duration of infertility (year) mean ± SD range	28.06 + 4.64 20 - 35   66.53 + 7.51 55 - 83   4.73 + 2.24 2 - 10	36.3 + 3.789 31 - 40   68.96 + 7.45 60 - 83

The total protein concentration in PF of unexplained infertility group (mean 27.4 + 4.96 mg/ml) was significantly higher than that in the PF of control group (mean 21.07 + 12.66 mg/ml) (P < 0.05).

Protein bands of PF samples detected by SDS- PAGE, from unexplained infertility and control groups were nearly similar. The mean number of protein bands in PF of the unexplained infertility was 8 (range 5-) bands and in the PF of fertile control group was 7 (range 5-8) bands. The molecular weight of the bands ranged from 130 kd to 17 kd. A protein band with molecular weight (MW) 32 kd was detected in the PF of 5/30 samples of unexplained infertility and not detected in any sample of fertile subjects. A protein band with MW 17 kd was detected in all cases of unexplained infertility group and in 7 cases of fertile control group.

Staining of the nitrocellulose membrane by non-specific colloidal gold revealed the same results of SDS-PAGE. By alkaline phosphatase immuno-blot kit the cytokine (TNF-a ) band appeared in PF of all cases of unexplained infertility, nearly at 17 kd and in 7 cases of fertile control group.

Embryotoxicity of PF of the unexplained infertility group was higher than that of fertile control group (P < 0.001). Embryos developed into blastocyst stage when 2-cell mouse embryos incubated with PF of unexplained infertile group and fertile control group were 220 / 310 and 280 / 300 embryos respectively (Table 2).

Table (2): The effect of peritoneal fluid of the unexplained infertility and infertile groups on the development of 2-cell mouse embryos

Data	Unexplained infertility	Fertile (Control)
* Total number of embryos * number of blastocysts * Percent of blastocysts Z P	310 220 70.96% 2.36 < 0.05	300 280 93.33%

Discussion

Data presented in this study indicated that women with unexplained infertility, previously thought to be normal by tradational tests (normal semen analysis, postcoital test, premenstrual endometrial biopsy, hysterosalpingography and diagnostic laparoscopy) demonstrated increase in protein concentration (mean 27.4± 4.69 mg/ml) of PF when compared with control group (mean 21.07± 12.66mg/ml) the variation was significant (P > 0.05). Analysis of protein in PF by SDS-PAGE revealed different protein bands in both PF of unexplained infertility (range; 5-11 bands) and fertile control (range; 5-8 bands) groups, molecular weight of the detected bands ranged from 130kd to 17kd. These findings are contradictory to other studies that reported no change in protein level in PF14,15.

In previous studies, analysis of PF protein of women with endometriosis and healthy control by native polyacrylamide gel electrophoresis showed undescribed protein with molecular weight 70kd in the secretory phase of all the peritoneal fluid samples (endometriosis and healthy control) and its absence during the proliferative phase14. On the other hand, Sueldo and associates (1987) did not detect this secretory protein by two-dimensional sodium-dodecyl sulphate gel electrophoresis. In the present study, PF was collected during the proliferative phase of the cycle. Protein content of PF varies from one phase to another during the cycle14.

In the presestudy, the detection of 32kd band in 5/30 patients of unexplained infertility group and its absence in all fertile control group may explain a hidden peritoneal cause of unexplained infertility. Previous investigators detected this band in all cases of endometriosis but not in healthy controls15.

Macrophages are attracted to the peritoneal environment more abundantly than any other cell types. Macrophages are capable of secreting various substances such as growth factors, cytokines, prostanoids, complements, and hydrolytic enzymes17. In our study, the cytokine (TNF-a ) was detected on nitrocellulose paper (by Western blotting) in all cases of unexplained infertility group and in only 5/30 cases of fertile control group. Rana and associates (1996) reported that TNF-a was synthesized at a greater level than normal by basal lipopolysaccharide stimulated macrophages from women with endometriosis. This level was also correlated with the level of TNF-a in PF, suggesting that the macrophages are the principal source of this cytokine in the PF.

Now, it is became evident that cytokines play an important role in reproduction at various levels including gamete function, fertilization, embryo development, implantation and post implantation survival of the conceptus19.

In the current study, the percentage of 2-cell mouse embryos that develop to blastocyst stage was 70.96% and 93.33% when incubated with PF of unexplained infertility and fertile control group respectively. So the peritoneal fluid samples of unexplained infertility group had an embryotoxic effect on the 2-cell mouse embryos than the control group. This result is in accordance with previous studies by Dodds et al., (1992) and Haney et al., (1994). PF of patients with unexplained infertility may have undiagnosed microscopic endometriosis22,23. Prough et al., (1990) noticed that PF from infertile patients with endometriosis during the follicular phase of the cycle did not promote 2-cell mouse embryos growth to the extent observed by fluid obtained from normal control. By ultrafiltration they separated 5 molecular weight fractions, all fractions inhibited mouse embryos growth to a great extent. They recorded that the molecular weight fractions >100kd showed greater inhibition to embryos development than did fractions < 100kd. This study suggested presence of humoral factors > 100kd that is inhibitory to mouse embryos development. These finding are contradictory to the present study in which protein fractions were separated by SDS-PAGE in the form of bands with variable molecular weight ranged from 17kd to 97kd.

Conclusion

The PF from patients with unexplained infertility contained high protein concentration, more protein bands, TNF-a band and exhibited more embryotoxic effect on 2-cell mouse embryos than the PF of fertile control group. These results may be one of the factors that explain the inability for conception in unexplained infertility. Further basic researches are needed to define the specific role of PF cells and soluble factors which may offer the possibility of more effective treatment of unexplained infertility. Immuno-regulatory therapy for such cases may be directed to the inhibition of lymphocytes proliferation and to normalize the plasma concentration of TNF-a and other cytokines as well as blocking the activation of macrophages.

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