Flow cytometric evaluation of leukocyte subpopulations in the follicular fluids of infertile patients.

OBJECTIVES: To evaluate the leukocyte subpopulations present in follicular fluid (FF) of infertile patients undergoing IVF-ET for tubal factor, idiopathic infertility, and endometriosis. PATIENTS: Sixty patients undergoing IVF-ET with a tubal factor diagnosis (n = 35), idiopathic infertility (n = 13), and endometriosis (n = 12) had their subpopulations of FF leukocytes analyzed by flow cytometry. MAIN OUTCOME MEASURE: Nonblood-contaminated samples of FF were collected under sterile conditions and centrifuged. Cells were labeled with a panel of monoclonal antibodies: anti-CD3, -CD4, -CD8, -CD14, -CD20, -CD45, and -CD56, and analyzed by cytofluorometry. RESULTS: Follicular fluid leukocytes from patients with idiopathic infertility had a significantly higher proportion of T lymphocytes than tubal factor and endometriosis patients. Endometriosis patients had significantly higher proportions of natural killer (NK) cells, B lymphocytes, and monocytes compared with groups of idiopathic infertility and tubal factor. CONCLUSIONS: The differences observed in the leukocyte subpopulations from FF of patients with idiopathic infertility and endometriosis may affect folliculogenesis and oocyte maturation. Moreover, these modifications could be one of the factors altering their fertility.

Endometrial T, B, and NK cells in patients with recurrent spontaneous abortion. Altered profile and pregnancy outcome.

Several lines of evidence indicate that immunologic effectors, particularly suppressor T cells and NK cells, may play a role in the pathogenesis of idiopathic repetitive abortions. To investigate the involvement of these immune cell populations, we determined the immunophenotypic characteristics of endometrial leukocytes from nonpregnant recurrent aborters. Habitual aborters with a negative investigation underwent an endometrial biopsy during their secretory phase and were followed prospectively to assess clinical outcome. Endometrial leukocytes were evaluated by two-color flow cytometric analysis. The percentage of endometrial CD8+ T lymphocytes was significantly decreased in recurrent aborters, and their CD4:CD8 ratio was increased. In contrast, the proportion of B lymphocytes (CD20+) was strikingly increased in these patients' endometria. The proportion of NK cells was identical in recurrent aborters and normal controls, but the CD16-CD56 bright NK cell subset, which is predominant in normal decidua and endometrium, was significantly decreased in favor of an important contingent of CD16+CD56 dim NK cells in all habitual aborters. Repetitive aborters who had normal CD8+ and CD20+ cell numbers and a normal CD4:CD8 ratio subsequently underwent successful pregnancies, while patients with continuing abortions presented lymphoid populations observed in the habitual aborters group. In conclusion, endometrial lymphocytes of recurrent spontaneous aborters harbor a distinct immunophenotypic profile that antedates implantation and suggests that endometrial immunologic conditions are intrinsically altered in recurrent aborters. Also, the prognostic impact of CD8 and CD20 expression supports their predominant role in the development of fetal tolerance. Finally, a role for NK cells in the abortion process is suggested by their altered subsets in all repetitive aborters.

Flow-cytometric characterization of hematopoietic cells in non-pregnant human endometrium.

PROBLEM: Immunologic evaluation and quantitation of hematopoietic cells in human endometrium has been difficult to perform, particularly in nonpregnant subjects. In this study, we describe a method for the flow-cytometric characterization of hematopoietic cells present in the endometrium of non-pregnant women. METHOD: Endometrial biopsy samples from normal donors were first mechanically disrupted and filtered to generate a single-cell suspension of leukocyte-enriched endometrial cells. Cells were labeled with a panel of monoclonal antibodies, stained with propidium iodide (PI), and one- or two-color flow-cytometric analysis performed on cells excluding PI. RESULTS: The methodology described in this study was highly reproducible in experiments evaluating the interrun and intrarun variability. We then determined the immunophenotypic profile of endometrial leukocytes from 12 normal females. The majority of leukocytes were T cells (CD3: 47%; CD4: 24%; CD8: 28%) with an important contingent of NK cells (CD56: 32%), the majority of which harbored the unusual CD16-CD56 bright phenotype, and a minority of B cells (CD20: 6%) and monocytes (CD14: 7%). CONCLUSIONS: Flow cytometry can be used to assess antigen expression on the surface of endometrial leukocytes from nonpregnant women. In future studies, it will be possible to use this approach to investigate the role of immune cell populations in the endometrium of patients experiencing reproductive failure.

Effect of follicular fluid supplementation on the in vitro development of human pre-embryos.

OBJECTIVE: To evaluate the value of supplementing a standard culture medium with 10% heat-inactivated mature follicular fluid (FF). DESIGN: Prospective randomized study evaluating the in vitro development of nontransferred, nonfrozen human pre-embryos in three culture conditions from day 3 to day 8 postfertilization. Preliminary evaluation by RIA and electrophoresis of factors responsible for these results. RESULTS: Ten percent mature FF supplementation of Inra Menezo (B2 medium) was associated with a significantly higher proportion of human pre-embryos reaching the morula (95% versus 72% with 10% maternal serum, P = 0.04) and the blastocyst stage (50% versus 11% with B2 alone, P = 0.02). The concentration of insulin-like growth factors I and II did not differ significantly between serum and mature FF-supplemented medium. Protein electrophoresis showed a difference of two bands corresponding to a molecular weight of 17,000 present in the serum and not in the FF-supplemented medium. CONCLUSION: Culture medium supplementation with 10% mature FF is associated with a significantly higher proportion of pre-embryos reaching the morula and blastocyst stage. The observed difference could be explained by the presence of a low molecular weight (17,000) embryotoxic factor contained in the serum-supplemented medium.

Embryonic resistance to tumour necrosis factor-alpha mediated cytotoxicity: novel mechanism underlying maternal immunological tolerance to the fetal allograft.

The cytokine tumour necrosis factor-alpha (TNF alpha) has been postulated to play an essential role in the cytotoxic activity of cell-mediated immunity against allogenic or tumour cells invading the host. Several tumour cell lines, however, are resistant to TNF mediated cytotoxicity and respond paradoxically by cellular proliferation and by autocrine secretion of TNF alpha. In view of the metastatic character of the mammalian embryo, the aim of this study was to assess the potential of murine embryos to secrete TNF alpha in vitro, to express TNF receptors and to resist TNF alpha mediated cytotoxicity during their in-vitro development to the blastocyst stage. The potential of human embryos to secrete TNF alpha in vitro until the blastocyst stage was also investigated. From a total of 11 human embryos, which were allowed to proceed to blastocyst formation, seven secreted TNF alpha in the range of 2-117 pg/ml/24 h. A total of 123 C57BL/6J mouse embryos were studied of which 55% secreted TNF alpha in the range of 1.25-3.95 mg/ml/24 h. The presence of high levels of exogenous TNF alpha (10-300 IU) was not detrimental to the in-vitro development of murine embryos. Using immunohistochemical techniques, we were not able to detect the presence of type I or II TNF receptors on the surface of murine embryos. Our findings suggest that human and C57BL/6J murine embryos have the potential to secrete TNF alpha in vitro during the developmental stages leading to blastocyst formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of lysoplatelet-activating factor on human sperm fertilizing ability.

OBJECTIVE: To evaluate the penetration rates in the hamster zona-free oocyte sperm penetration assay (SPA) after exposure of spermatozoa to lysoplatelet-activating factor (LPAF) and lysophosphatidyl choline (LPC). DESIGN: Washed human spermatozoa were exposed to 100 microM of LPAF or LPC, followed by the assessment of their fertilizing ability using the SPA. The percentage of penetration, the sperm binding in the SPA, the percentage of motile spermatozoa, and the acrosome reaction rates were quantified. SETTING: Private research and university laboratories. PATIENTS, PARTICIPANTS: Fresh and frozen semen samples from fertile donors with proven fertility were used as well as fresh semen from infertile patients attending a fertility clinic. All the infertile patients had abnormal semen analysis. INTERVENTIONS: Human spermatozoa were incubated for 90 minutes in the presence or absence of LPAF or LPC at 100 microM with 0.3% albumin in Ham's F-10 (GIBCO, Dorval, Quebec, Canada), and their fertilizing ability was evaluated using the SPA. The effect of these lysophospholipids on the percentage of acrosome reaction was evaluated with a fluorescent microscopy technique. RESULTS: The penetration rates of the SPA in male factor increased significantly from 3% +/- 6% with controls to 19% +/- 9% and 34% +/- 22% after incubation with LPC and LPAF, respectively. Sperm-oocyte binding was not significantly increased in this group. Sperm penetration assay penetration rates were also increased in fertile cryopreserved spermatozoa with LPC and LPAF. In this group, the acrosome reaction was significantly increased from 2% +/- 1% in controls to 10% +/- 6% and 8% +/- 3% after incubation with LPC and LPAF, respectively. CONCLUSION: Lysoplatelet-activating factor and LPC independently increased the penetration rate of spermatozoa and the percentage of acrosome reaction. Lysophosphatidylcholine and LPAF may be beneficial in the treatment of spermatozoa with male factor infertility and may increase fertilization rates in IVF.