bluuebunny/crossref_metadata_2025 huggingface dataset:
Pregnancy‐Associated Uterine Granulated Metrial Gland Cells in Mutant and Transgenic Mice
Authors: Guimond Marie‐Josée , Wang Baoping , Fujita Jun , Terhorst Cox , Croy B. Anne
Published: September 2011

PROBLEM: Granulated metrial gland (GMG) cells are pregnancy‐specific uterine lymphocytes found in rodents. The lineage relationships of GMG cells are incompletely defined, although genetic and immunophenotyping studies suggest membership in the natural killer (NIC) cell lineage. Pregnancy‐specific functions have been postulated for GMG cells, but no successful depletion of these cells has been achieved that would permit assessment of any critical roles that might influence gestational outcome.

METHOD: Routine histological methods for light microscopy were used to assess implantation sites from wild‐type mice and mice of the following genotypes: tgE26, mi/mi, and p53 knockout.

RESULTS: GMG cells are transient, histamine‐negative cells found in the pregnant uteri of most mice. Pregnancies in the NK and T‐cell‐deficient tgE26 were characterized by infrequent GMG cells, very small placentae, and an elevated rate of fetal and perinatal mortality. In term placentae of mi/mi with dystocia, GMG cells were found in a new location along the plane of placental separation, and they appeared degranulated. A normal life‐history was observed for GMG cells in p53 knockout mice.

CONCLUSION: Pregnancies in mutant and transgenic mice indicate that GMG cells are natural killer cells and have critical roles in placental development and pregnancy success. The disappearance of GMG cells at term is independent of p53 gene expression.
Inhibition of Macrophage Nitric‐Oxide Production and Ia‐Expression by Docosahexaenoic Acid, a Constituent of Fetal and Neonatal Serum
Authors: Khair‐El‐Din Tarik A. , Sicher Stanley C. , Vazquez Miguel A. , Lu Christopher Y.
Published: September 2011

PROBLEM: We previously demonstrated profound inhibition of macrophage activation in the murine placenta in vivo. Given the importance of macrophages both in initiating cellular immunity by presenting antigen in the context of Ia to CD4+ T cells, and in killing cellular targets by producing nitric oxide (NO), inhibition of macrophage functions in the placenta may account for the increased susceptibility of the placenta to infection. We have also showed that docosahexaenoic acid (DHA), at concentrations present in the fetal circulation, has a major role in inhibiting macrophage Ia‐expression and NO production in the placenta. The concentration of DHA in fetal serum perfusing the placenta is 50x higher than in the adult. DHA has previously been reported to profoundly affect prostanoid production, to be metabolized by lipoxygenases, and to affect lipoxygenases. We now determine if these activities of DHA account for its inhibition of macrophage NO production and Ia‐expression. METHODS: Murine macrophages were cultured in vitro, exposed to IFNγ, endotoxin, DHA, and various eicosanoids, and their ability to produce NO or express Ia determined.

RESULTS: Although the cyclooxygenase inhibitor, indomethacin, did inhibit NO production, DHA inhibited by a different mechanism. DHA further inhibited NO production by macrophages exposed to doses of indomethacin known to maximally inhibit prostanoid production. Stable, biologically active prostanoids did not reverse the inhibitory effect of DHA. Although DHA is metabolized by lipoxygenases, the lipoxygenase inhibitor NDGA did not reverse the inhibition of either NO production nor Ia expression. This indicates that lipoxygenase products of DHA did not mediate inhibition. NDGA itself inhibited NO production and Ia expression. However, DHA did not inhibit by inhibiting lipoxygenase activity because DHA further inhibited macrophages exposed to doses of DHA known to maximally inhibit lipoxygenases. Furthermore, stable biologically active analogs of lipoxygenase products did not reverse DHA inhibition. DHA also did not inhibit by preventing PAF production because PAF did not reverse inhibition of NO production.

CONCLUSION: DHA did not inhibit Ia‐expression or NO production via its known effects on eicosanoid or PAF metabolism, nor by being metabolized by lipoxygenases.
Quantitative Assessment of Cytokines (GROα and IL‐10) in Human Seminal Plasma During Genitourinary Inflammation
Authors: Rajasekaran Mahadevan , Hellstrom Wayne , Sikka Suresh
Published: September 2011

PROBLEM: Mechanisms involved in infertility due to genitourinary (GU) inflammation are unknown. The production of pro‐inflammatory (GROα) and anti‐inflammatory (IL‐10) cytokines in seminal plasma is monitored in this study.

METHOD: GROα, IL‐10, and granulocyte elastase were evaluated in semen from I) normal, II) infertile patients, and III) infertile patients with leukocytospermia.

RESULTS: GROα in infertile patients with GU inflammation was 1.5‐fold higher compared to group II and 2.5‐fold higher compared to group I patients. The IL‐10 levels were 12‐ to 16‐fold lower compared to GROα in these samples. Mean ratio of GROα to IL‐10 was higher in group III than the other two groups. A positive correlation was observed between granulocyte elastase and GROα in all groups. Group III patients exhibited poor sperm parameters.

CONCLUSIONS: A shift towards increased production of pro‐inflammatory chemokine GROα may have a potential role in male infertility associated with GU inflammation.
Elevated Nitric Oxide Concentration in the Seminal Plasma of Infertile Males: Nitric Oxide Inhibits Sperm Motility
Authors: Nobunaga Toshikatsu , Tokugawa Yoshihiro , Hashimoto Kazumasa , Kubota Yasue , Sawai Keisuke , Kimura Tadashi , Shimoya Koichiro , Takemura Masahiko , Matsuzaki Noboru , Azuma Chihiro , Saji Fumitaka
Published: September 2011

PROBLEM: To evaluate the “effect of nitric oxide in the seminal plasma on sperm motility. METHOD: Seminal plasma concentrations of NO2—, a stable end product of nitric oxide, of 108 males of infertile couples and 15 proven fertile donors were measured and compared with spermatogram parameters. Motile sperm was incubated with a nitric oxide‐generating drug, sodium nitroprusside, for 6 hr in the absence or presence of oxyhemoglobin, an inhibitor of nitric oxide.

RESULTS: The NO2— concentration in the seminal plasma was 6.58±0.56 μM in 26 infertile males with leukocytospermia, 5.51±0.25 μM in 82 infertile males without leukocytospermia, and 3.91±0.16 μM in 15 controls. There was a significant correlation between the NO2— concentration and sperm motility. Sodium nitroprusside reduced the sperm motility in a dose‐ and time‐dependent manner and its reduction was completely inhibited by the addition of oxyhemoglobin.

CONCLUSION: These findings indicate that nitric oxide concentration in the seminal plasma of infertile males is elevated and that nitric oxide is an inhibitor of sperm motion.
Estrogen Blocks Early T Cell Development in the Thymus
Authors: Rijhsinghani Asha G. , Thompson Kristin , Bhatia Sudershan K. , Waldschmidt Thomas J.
Published: September 2011

PROBLEM: Pregnancy and estrogen are known to suppress B lymphopoiesis as well as lead to thymic involution in the mouse. Additionally, estrogen deficiency by oophorectomy reportedly causes a selective increase in the B220+ B cells in the murine bone marrow. The purpose of this study was to determine if estrogens played a regulatory role in T cell development.

METHODS: The first experimental group consisted of 5–6‐week‐old Balb/c mice that received subcutaneous pellets of placebo, estriol, estradiol, or progesterone. The thymus glands were examined 2–4 weeks after treatment. The second group consisted of 6‐week‐old Balb/c mice who underwent either bilateral oophorectomy or a sham procedure. Two weeks after the surgery, extensive phenotypic characterization of the thymus and spleen cells was performed by flow cytometry using monoclonal antibodies to surface markers of T cell subsets.

RESULTS: Estrogen treatment causes a dramatic reduction of thymic size and cellularity. All defined T cell subsets of CD4 and CD8 were reduced, with a disproportionate loss of CD4+CD8+ double positive cells. Examination of the triple negative (CD3‐CD4‐CD8‐) subset revealed a striking loss of TN developmental progression of the early precursor cells. Based on the expression of CD44 (pgp‐1) and CD25 (IL‐2Rα) markers, the TN thymic compartment was composed almost entirely of the earliest population (CD44+, CD25‐), with the remaining maturational stages (CD44+, CD25+; CD44‐, CD25+; CD44‐, CD25‐) depleted. In contrast, all T cell developmental stages in the thymus were found to be in normal proportions in the oophorectomized mice, with no differences in the splenic T and B cell subsets.

CONCLUSIONS: The study demonstrates that estrogen but not progesterone blocks T cell development in the thymus. However, contrary to our expectation, estrogen deprivation by oophorectomy does not enhance T cell development.
Modulation of NK Cell Lytic Function by Endometrial Secretory Factors: Potential Role in Endometriosis
Authors: Somigliana Edgardo , Viganò Paola , Gaffuri Barbara , Candiani Massimo , Busacca Mauro , Blasio Anna Maria Di , Vignali Mario
Published: September 2011

PROBLEM: Development and progression of endometriosis have been suggested to be related to a defect of NK cells in the ability to eliminate endometrial fragments regurgitated with menstrual debris in ectopic sites. However, it has not been clarified yet whether the origin of this defect has to be attributed to an intrinsic alteration of NK cell population or to a consequence of the environmental system. The present study was designed to evaluate whether soluble factors of endometrial origin might directly interfere with NK cell lytic function.

METHOD: Media conditioned by 20 endometrial stromal cultures (10 from endometriosis patients and 10 from women without the disease) were examined to test their effects on NK cell‐mediated cytotoxicity directed against endometrial stromal targets. NK cells were purified from peripheral blood samples by a two steps panning technique and incubated overnight with and without endometrial supernatants. Thereafter, the percentage of lysis against endometrial cells was detected in a 51Cr cytotoxicity assay.

RESULTS: Media conditioned by the overall endometrial stromal cultures exerted a significant suppressive effect (P < 0.001) on NK cell function when compared to control NK fractions (mean percentage of cytotoxicity ± SEM 17.46 ± 2.8 and 29.41 ± 1.49, respectively). No inhibitory effect was detect with conditioned media obtained from peritoneal cells and keratinocytes. Moreover, NK cell‐mediated cytotoxicity resulted significantly more inhibited by the addition of endometrial supernatants obtained from endometriosis patients (mean percentage cytotoxicity ± SEM 13.55 ± 2.3, P < 0.05) when compared to those from women without the disease (mean percentage cytotoxicity ± SEM 21.38 ± 4.9).

CONCLUSION: The results of this study indicate the existence of soluble non‐specific factor(s) released by human endometrial cells able to interfere with NK cell killing. These factors could participate in the local regulation of the immunological mechanisms suggested to be involved in the development of endometriosis.
Psycho‐Neuro‐Cytokine/Endocrine Pathways in Immunoregulation During Pregnancy
Authors: Clark David A. , Arck Petra C. , Jalali Reza , Merali Fatima S. , Manuel Justin , Chaouat Gerard , Underwood Jennifer L. , Mowbray James F.
Published: September 2011

PROBLEM: Some mammalian pregnancy failure is thought to occur by immunological or immunologically modifiable mechanisms. The original model wherein spontaneous abortion was proposed to represent rejection of the conceptus as an allograft has been supplanted by a model of maternal paraimmunological natural effector cell toxicity to fetal trophoblast more closely related to tumor rejection. The problem is to integrate current information concerning the role of immunological, paraimmunological, endocrinological, and stress‐triggered neural factors that determine whether or not abortion will occur.

METHODS: Review of existing data.

RESULTS: An integrated model is proposed.

CONCLUSION: Immunological factors play an important role in abortion processes and prevention of abortions. The existence of abortogenic mechanisms and their regulation appears to be based upon optimizing survival of the species. Two new conceptual models provide a useful framework for further investigation of human pregnancy failure and its treatment.
Transforming Growth Factor‐β2‐Related‐Decidual Suppressor Factor Is Not Related to TJ6 Protein
Authors: Merali Fatima S. , Arck Petra C. , Beaman Kenneth , Clark David A.
Published: September 2011

Transforming growth factor (TGF)‐β2‐related‐decidual suppressor factor (DSF) and TJ6 protein are both immunosuppressive molecules present in murine and human pregnancy. Treatment of mice with either anti‐TJ6 or anti‐TGF‐β2 neutralizing antibodies results in increased fetal loss. Western blots of supernatants from pregnant mouse decidua probed with anti‐TJ6 (soluble form) showed a doublet at a similar molecular size as when the blot was probed with anti‐TGF‐β2 antibody. The problem is to determine whether TJ6 and DSF are the same protein. In order to determine if TJ6 and DSF are the same or different proteins, we used affinity column purified TGF‐β2‐DSF and stained Western blots with anti‐TJ6. The TGF‐β2‐monoclonal antibody affinity column‐purified DSF that stained with anti TGF‐β2 was not reactive with anti‐TJ6 antibody. TJ6 has only a 30% gene sequence homology and a 13% amino acid homology to TGF‐β2. TJ6 and TGF‐β2‐related DSF appear to be different immunosuppressive proteins in decidua.
Analyses of HLA‐DQ Alleles in Recurrent Spontaneous Abortion (RSA) Couples
Authors: Wagenknecht Dawn R. , Green Karen M. , McIntyre John A.
Published: September 2011

PROBLEM: The influence of HLA sharing on pregnancy outcome is controversial. In renal transplantation, HLA‐DQB1 donor‐recipient mismatches have been shown beneficial for long‐term transplant success. Since pregnancy is defined as Nature's allograft, we investigated the relevance of HLA‐DQ mismatching in normal reproducing couples compared to couples experiencing RSA.

METHOD: Unexplained RSA couples referred to our laboratory for immunological testing were classified by immunological findings and obstetrical history. Primary RSA couples shared ≥ 2 HLA‐A, B, or DR antigens, had no cytotoxic anti‐paternal antibodies, and no gestation beyond 20 weeks. Secondary RSA couples had cytotoxic anti‐paternal antibodies and RSA after a live birth. HLA‐DQA1 and DQB1 alleles were identified by PCR‐SSP.

RESULTS: No differences in DQA1 and DQB1 mismatch were observed among RSA patients and controls. DQA1‐DQB1 haplotype mismatches were not different among the three groups of couples.

CONCLUSIONS: In contrast to renal transplant, HLA‐DQ incompatibility did not differ among RSA couples compared with successful reproducing couples.
Angiogenesis and the Expression of Vascular Endothelial Growth Factor in Endometrium and Placenta
Authors: Torry Donald S. , Torry Ronald J.
Published: September 2011

PROBLEM: The demand for increased angiogenesis and microvascular permeability during cyclical changes in the endometrium and during placentation raises the possibility that aberrations in these events could lead to suboptimal reproductive performance. However, relatively little is presently known regarding the regulation of vascular growth and permeability in these tissues.

METHOD OF STUDY: This review of current literature focuses on the expression, regulation, and potential physiological effects of vascular endothelial growth factor (VEGF) within endometrial and placental tissue.

RESULTS: Spatial and temporal expression of VEGF as well as its restricted specificity, essential role in vasculogenesis/angiogenesis, and ability to induce vascular permeability makes VEGF an attractive regulator of vascular growth and permeability in the endometrium and placenta.

CONCLUSION: A better understanding of the production, regulation, and physiological responses of the vasculature to angiogenic growth factors may lead to new therapeutic strategies for reproductive disturbances secondary to vascular insufficiencies within the female reproductive tract.
Review: Immunobiology of Preeclampsia
Authors: Taylor Robert N.
Published: September 2011

Preeclampsia has been recognized clinically since the time of Hippocrates: however its etiology and pathophysiology remain enigmatic. This pregnancy‐specific syndrome typically presents in late pregnancy as hypertension, edema, and proteinuria. Investigations over the past 15 years have revealed that preeclampsia is associated with abnormal placentation, reduced placental perfusion, endothelial cell dysfunction, and systemic vasospasm. Since it occurs more commonly in Primigravidae and in women with underlying collagen‐vascular diseases, an immunological component has long been suspected. Increased prevalence in high‐order and molar pregnancies and those associated with increased placental mass suggests that trophoblastic volume and fetal antigen load are correlated with the syndrome. Epidemiological reports indicate that the prevalence of preeclampsia is decreased in women who received heterologous blood transfusions, practiced oral sex, or when a long period of cohabitation preceded an established pregnancy. Conversely, the use of condoms as a primary mode of contraception is associated with a higher risk of preeclampsia. These studies suggest that prior exposure to foreign or paternal antigens imparts a protection against the likelihood of developing preeclampsia. Clinical evidence of cellular and humoral immune dysfunction is associated with the syndrome. Fibrin and complement deposition and “foam” cells in atherosis lesions resemble the histopathology of renal allograft rejection. Relative T‐cell, natural killer cell, and neutrophil activation have been reported in preeclampsia and circulating cytokines and antiphospholipid antibodies are more prevalent in preeclamptic than in normal pregnant women. These abnormalities are consistent with the systemic endothelial cell dysfunction that has been postulated as a pathophysiological feature of preeclampsia. While such associations do not prove causality, they suggest testable hypotheses for continued basic and clinical investigation of this major complication of human pregnancy.
The Role of Trophoblast Interferons in the Maintenance of Early Pregnancy in Ruminants
Authors: Godkin James D. , Smith Scott E. , Johnson Roger D. , Doré Jules J.E.
Published: September 2011

PROBLEM: Are the effects of ruminant trophoblast interferon‐tau (IFN‐τ) on uterine prostaglandin (PG) secretion a specific action of this cytokine and what are the effects of IFN‐τ on expression of uterine genes not generally associated with pregnancy maintenance?

METHODS: The effects of IFN‐τ and IFN‐α on bovine uterine expiant and epithelial cell production of PGF2α and PGE2 were determined in the presence and absence of oxytocin (OT). The effects of intrauterine administration of IFN‐τ were determined on uterine expression of retinol‐binding protein (RBP) and transforming growth factor‐beta (TGF‐β) isoforms.

RESULTS: IFN‐τ attenuated uterine endometrial secretion of PGF2α and PGE2 in vitro and diminish PG stimulation by OT. IFN‐τ and IFN‐α were observed to be equipotent. Intrauterine infusion of IFN‐τ resulted in a significant decrease in steady‐state RBP mRNA levels and expression of TGF‐B1, 2, and 3 mRNA levels were lowest in IFN‐τ treated animals.

CONCLUSION: Negative regulation of gene expression may be a general strategy in IFN activity. This may explain the similar activities of IFN‐τ and IFN‐α on a broad variety of cell types, including ruminant uterine endometrium.
Elevated Amniotic Fluid Interleukin‐6 Levels During the Early Second Trimester Are Associated With Greater Risk of Subsequent Preterm Delivery
Authors: GHIDINI ALESSANDRO , JENKINS CHUKA B. , SPONG CATHERINE Y. , PEZZULLO JOHN C. , SALAFIA CAROLYN M. , EGLINTON GARY S.
Published: September 2011

PROBLEM: Subclinical intra‐amniotic infection is often associated with preterm delivery and may precede it by several weeks. We tested the hypothesis that Interleukin‐6 (IL‐6) may be elevated in the midtrimester amniotic fluid of pregnancies destined to deliver preterm.

METHODS: A historical cohort study was designed to compare the amniotic fluid (AF) concentrations of IL‐6 at 14–20 weeks in a group of women subsequently delivering at ≤ 34 weeks (n = 13) with those of women delivering at term (n = 166). Included were singleton gestations with no evidence of fetal structural or chromosomal abnormalities, or maternal conditions known to be associated with preterm delivery (n = 179). Levels of IL‐6 were measured by immunoassay and correlated with demographic and pregnancy outcome information. Statistical analysis included correlation, one‐way ANOVA after log‐transformation, contingency tables, logistic regression, and receiver operator characteristic (ROC) curve analysis.

RESULTS: There was an inverse correlation between AF IL‐6 levels at 15–20 weeks and gestational age at delivery (r = −0.16, P = 0.03). Women delivering at ≤ 34 weeks had significantly higher median AF IL‐6 levels (570 pg/ml versus 330 pg/ml, P < 0.0001), rate of African American race (50% versus 12%, P = 0.004), and of infants with birth weights < 10th centile (31% versus 7%, P = 0.02) than women delivering at ≥ 37 weeks. Logistic regression analysis showed that IL‐6 was independently associated with PTD at ≤ 34 weeks after controlling for race and birth weight centiles (P = 0.039).

CONCLUSIONS: AF IL‐6 at 15–20 weeks can identify patients at risk for PTD at ≤ 34 weeks, suggesting that a portion of PTD cases have inciting events that take place during the early second trimester.

CAPSULE: Midtrimester amniotic fluid IL‐6 concentrations are significantly higher in women subsequently delivering preterm at ≤34 weeks compared with those delivering at term.
Inhibition of Interleukin‐10 During Pregnancy Results in Neonatal Growth Retardation
Authors: RIJHSINGHANI ASHA G. , THOMPSON KRISTIN , TYGRETTE LORRAINE , BHATIA SUDERSHAN K.
Published: September 2011

PROBLEM: Interleukin 10 is considered to be important in the survival of the fetus in murine pregnancies that are known to be at risk for fetal wastage. The function of IL‐10 in a normal pregnancy is not known.

METHODS: In this report, we attempted to neutralize Interleukin 10 by administering anti IL‐10 monoclonal antibodies (mAb) to pregnant mice that have a low background risk for fetal resorptions. The first group of mice was sacrificed on gestation day 18 to study the fetal effects of anti IL‐10 administration. The second group of mice was allowed to deliver to study the effects on the neonatal outcome.

RESULTS: Administration of anti IL‐10 mAb did not affect the duration of gestation or the fetal outcome. Neonates exposed to anti IL‐10 mAb in utero showed signs of transient growth deficiency starting at 4 weeks of age that spontaneously corrected by 6 weeks of age.

CONCLUSIONS: Administration of anti IL‐10 mAb does not alter the duration of gestation or the fetal outcome in normal murine pregnancies; however, it appears to be associated with transient neonatal growth problems.
Monoclonal Antibodies as Direct Probes for Human Sperm Acrosome Reaction
Authors: DORJEE S. , LAI C.L.B. , LEE A. , LEE C.Y.G
Published: September 2011

PROBLEM: To develop simple and rapid assay procedures for determining human sperm acrosome reaction under various experimental conditions.

METHODS: Specific monoclonal antibodies against human sperm acrosome were generated and utilized as probes for acrosome reaction assays by direct labelling of antibodies with fluorescein‐isothiocyanate (FITC).

RESULTS: Among the generated monoclonal antibodies, HS‐63 was shown to react with antigens in the acrosome content of permeabilized acrosome‐intact human sperm, but not with those of the live ones. HSA‐10 was found to recognize antigens on the surface of sperm inner acrosome and to react with acrosome‐reacted human sperm in suspension. Following a swim‐up procedure, highly motile sperm were recovered and incubated in BWW medium at 37°C for 18 h. The percentages of acrosome‐reacted sperm were determined at various incubation times by using FITC‐labeled HS‐63 or HSA‐10 as the indicators in 10 min direct immunofluorescent assay. With the FITC‐labeled HS‐63 probe, the percentage of positively stained human sperm fixed in methanol decreased significantly after 18 h of incubation from >90 to 70–80%. In contrast, positively stained live human sperm by HSA‐10 increased significantly from <1% to 15–30%. A high correlation was obtained between the use of monoclonal antibodies and PSA (Pissum sativumagglutinin) for direct assessment of human sperm acrosome reaction.

CONCLUSION: In view of the simplicity in assay procedures and evaluations, these FITC‐labeled monoclonal antibodies are reliable probes for direct assessment of acrosomal status of human sperm.
Acrobeads Test as a Predictor of Fertilization In Vitro
Authors: HERSHLAG AVNER , PAINE TERRY , SCHOLL GERALD M. , ROSENFELD DAVID L. , ZHU JING ZHANG , GUHRING PATRICIA , MECEROD DEBORAH , BENOFF SUSAN , MANDEL FRANCINE S. , BENOFF SUSAN
Published: September 2011

PROBLEM: To determine whether the results of the Acrobeads test, which measures the expression of the complement regulator molecule CD46 on the inner acrosomal membrane following the acrosome reaction, accurately identifies semen specimens that will exhibit reduced or failed fertilization following conventional IVF insemination.

METHOD: The Acrobeads test was performed on semen specimens from 97 consecutive patients preparing to undergo an PVF cycle utilizing a standardized insemination protocol. Motile sperm populations were examined at 6 h and 24 h post‐isolation for sperm‐bead agglutination. Results of the Acrobeads test were compared to that of TRITC‐PSA staining in matched specimens to directly measure the spontaneous loss of acrosome content. The percentages of TRITC‐PSA‐negative sperm were determined in freshly isolated motile populations and in duplicate aliquots incubated 18 to 20 h under sperm capacitating conditions. The relationship between the results of both analyses estimating spontaneous acrosome reactions and the rate of fertilization of metaphase II oocytes was examined.

RESULTS: The Acrobeads score did not correlate significantly with the rate of fertilization by insemination at 6 h or at 24 h. The negative predictive value of this test was 21.4%. There was no correlation between the Acrobeads score and the percentage of sperm undergoing a spontaneous acrosome reaction as detected by TRITC‐PSA labeling. In contrast, the increment increase in the percentage of spontaneous acrosome reactions as quantified by TRITC‐PSA staining was correlated with the fertilization rate.

CONCLUSIONS: Contrary to previous reports, our prospective, double‐blinded study failed to demonstrate that the Acrobeads test can accurately predict fertilization outcome in IVF. Therefore, the routine use of this test to screen patients prior to an IVF cycle in order to select appropriate treatment (i.e., ICSI) cannot be recommended.
Changes in T, B, and NK Lymphocyte Subsets During and After Normal Pregnancy
Authors: Watanabe Mikio , Iwatani Yoshinori , Kaneda Tatsunari , Hidaka Yoh , Mitsuda Nobuaki , Morimoto Yasuhiko , Amino Nobuyuki
Published: September 2011

PROBLEM: Pregnancy affects the maternal immune system and the clinical course of maternal diseases. Here we report the changes in the detailed lymphocyte subsets of helper T cells, suppressor T cells, CD5+ B cells, T cell receptor (TCR) αβ‐positive T cells (Tαβ cells), TCRαβ‐negative T cell (Tγδ cells), and others during and after pregnancy through to one year postpartum, and discuss the significance of the changes.

METHOD: The absolute numbers of helper T cells, suppressor T cells, cytotoxic T cells, TCRαβ‐negative T cells (Tγδ cells), CD5— B cells, CD5+ B cells, and NK cell subsets were examined by two‐color flow cytometry in peripheral blood from 51 healthy non‐pregnant women, 106 healthy pregnant women, and 148 healthy postpartum women.

RESULTS: In early pregnancy, the numbers of suppressor T cells and NK cells with strong cytotoxicity (NK+++ cells) increased, and the number of cytotoxic T cells decreased. In late pregnancy, the helper T cell and NK+++ cell numbers decreased. Tαβ, CD5— B and CD5+ B cells decreased during pregnancy. After delivery, helper T cells and cytotoxic T cells increased from 1 to 4 months postpartum, and suppressor T cells increased at 7 months postpartum. TCRαβ‐negative T cells increased at 4 to 10 months postpartum. Both CD5— and CD5+ B cells decreased further at 1 month postpartum, but CD5+ B cells increased markedly at 7 to 10 months postpartum.

CONCLUSIONS: These data indicate that 1) early increases of suppressor T cells and NK+++ cells during pregnancy may be related to the mechanism to accept or reject the fetus in early pregnancy, respectively; 2) late decreases of helper T cells and NK+++ cells may be related to the maintenance of pregnancy: 3) postpartum increases of helper T cells, cytotoxic T cells, TCRαβ‐negative T cells (Tγδ cells), and CD5+ B cells may be related to the postpartum aggravation of autoimmune diseases; and 4) the immunological effects of pregnancy remains until about 1 year after delivery.
Low Serum M‐CSF Levels Are Associated with Unexplained Recurrent Abortion
Authors: Katano K. , Matsumoto Y. , Ogasawara M. , Aoyama T. , Ozaki Y. , Kajiura S. , Aoki K.
Published: September 2011

PROBLEM: The purpose of this study was to determine whether the serum macrophage‐colony stimulating factor (M‐CSF) level is associated with early pregnancy loss in unexplained recurrent spontaneous abortion (RSA) patients.

METHOD: We therefore compared preconceptional serum M‐CSF levels between unexplained RSA patients and controls. The former comprised 44 bed‐rest therapy patients and 43 intradermal immunization (IDI) patients receiving paternal lymphocyte therapy, who had experienced two and three or more consecutive first‐trimester pregnancy losses, respectively. The controls were 46 healthy non‐pregnant women. We also prospectively studied the association between M‐CSF levels during pregnancy and adverse pregnancy outcomes. Sera from a total of 31 pregnant women, including 16 of the bed‐rest therapy group and 15 of the IDI therapy group, were collected at the 4th, 6th, and 8th gestational weeks and were measured for M‐CSF levels, using the enzyme‐linked immunoadsorbent assay (ELISA) method established by Hanamura et al.

RESULTS: Serum M‐CSF levels were significantly lower in the non‐pregnant RSA patients (460.0 ± 185.6 U/ml; mean ± SD) than in the control group (726.5 ± 134.0 U/ml) and also were lower at the 8th, but not the 4th or 6th gestational week in those patients of both the bed‐rest and IDI therapy groups whose outcome was pregnancy failure.

CONCLUSION: Thus a low level of serum M‐CSF was found to be associated with unexplained recurrent pregnancy loss in both the preconceptional and conceptional phases. These results raise the possibility that M‐CSF may play an important role in the maintenance of pregnancy and that it can be used as a parameter for determining individuals at risk.
Identification of Epitopes of Monoclonal Antibodies to Porcine Zona Pellucida 3β Glycoprotein, a Homologue of the Mouse/Human Sperm Receptor
Authors: Afzalpurkar Abhijit , Gupta S.K.
Published: September 2011

PROBLEM: Immunization with zona pellucida (ZP) glycoproteins leads to a block in fertility with a variable degree of ovarian dysfunction. To avoid autoimmmune oophoritis, synthetic peptides corresponding to B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. The main objective of the present study is to define the epitopes recognized by monoclonal antibodies (mAbs) generated against porcine ZP3β, a homologue of the designated primary sperm receptor in mice and humans.

METHODS: A multipin synthetic peptides approach has been used to map the epitopes recognized by mAbs. Dodecapeptides with an overlap of 6 amino acids corresponding to a precursor pZP3β‐deduced amino acid sequence (excluding the signal sequence) were synthesized on polypropylene pins and were tested for their reactivity with mAbs by enzyme‐linked immunoadsorbent assay (ELISA). The ability of synthetic peptides corresponding to the identified epitopes to inhibit the binding of mAbs to pZP3β in a competitive inhibition ELISA was investigated to confirm the above findings.

RESULTS: Reactivity of the mAbs with the pin‐bound peptides in ELISA‐identified epitopes for mAb‐451 to EEKLVF (166–171) and mAb‐462/470 to FKAPRP (250–255) amino acid residues. mAb‐30 recognized QPVWQDEGQRLR (23–34) and VICRCC (316–321) amino acid residues. Competitive inhibition with synthetic peptides encompassing the motifs corresponding to 23–34 and 316–321 for binding of mAb‐30 to pZP3β revealed the epitopic domain to be 23–34 amino acids. Synthesis of overlapping octapeptides further identified WQDE as the minimum motif for binding of mAb‐30, and the replacement of one amino acid at a time with glycine revealed tryptophan as the critical residue.

CONCLUSIONS: Collectively, these results describe peptide epitopes that will help in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZP3β or its homologues in other species.
T‐Helper 2 Type Cytokine and Soluble Interleukin‐2 Receptor Levels in Seminal Plasma of Infertile Men
Authors: Paradisi Roberto , Mancini Rita , Bellavia Emanuela , Beltrandi Elisabetta , Pession Annalisa , Venturoli Stefano , Flamigni Carlo
Published: September 2011

PROBLEM: The role of cell‐mediated immunity (CMI) in unexplained male infertility and impaired sperm function has been explored.

METHOD OF STUDY: The presence of cytokines, namely, interleukin‐4 (IL‐4), interleukin‐6 (IL‐6), and the soluble interleukin‐2 receptor (SIL‐2R), was investigated in seminal plasma of 18 fertile and 20 infertile subjects, using specific enzyme‐linked immunoadsorbent assays. RESULTS: IL‐4 was not detected. SIL‐2R was detected, but the concentration difference between the fertile and infertile group was not significant. IL‐6 was detected with significantly higher levels in the infertile group compared to the fertile group. IL‐6 levels in seminal plasma correlated positively with leukocyte count and negatively with sperm count, motility, and morphology.

CONCLUSIONS: These findings show: a) a lack of IL‐4 in seminal plasma; b) similar SIL‐2R levels in fertile and infertile seminal plasma; c) increased IL‐6 secretion in seminal plasma of infertile subjects; and d) specific correlations of IL‐6 with the main semen parameters.
The Anovulation in Female Mice Resulting from Postnatal Injections of Estrogen is Correlated with Altered Levels of CD8+ Lymphocytes
Authors: Deshpande Rohini R. , Chapman John C. , Michael Sandra D.
Published: September 2011

PROBLEM: Injections of estradiol‐17β (E2) are known to both induce anovulation and alter lymphocyte maturation in female mice. The current study examined whether the two events are related.

METHOD OF STUDY: Female (C3H/HeJ × 129J)F1 (C31) mice were injected with 20 μg of E2 from 0–3 days, or from 3–6 days, postpartum. At 8, 12, 20, 32, or 40 weeks of age, the animals were killed, T lymphocytes were characterized, and ovaries were histologically examined for the presence of corpora lutea.

RESULTS: Animals injected with E2 from 0–3 days postpartum had percentages of CD8+ thymocytes and CD8+ splenocytes that were always lower than in noninjected females, and the E2‐injected animals never ovulated, even by 40 weeks of age. In contrast, animals injected with E2 from 3–6 days of age had percentages of CD8+ thymocytes and CD8+ splenocytes that, although initially lower than in control females, attained control values by 32 weeks of age. In addition, at 32 weeks of age a number of the 3–6‐day E2‐injected females ovulated, whereas at earlier ages none had. Further, injections of E2 had little effect on the percentages of CD4+ thymocytes and splenocytes in these animals.

CONCLUSIONS: The results suggest that E2‐induced anovulation in C31 female mice is correlated with decreased levels of CD8+ lymphocytes, and an increased CD4+/CD8+ lymphocyte ratio.
The Regulatory Role of Transforming Growth Factor‐Beta in Activation of Milk Mononuclear Cells
Authors: Ayoub I.A. , Yang T.J.
Published: September 2011

PROBLEM: Immunoregulatory role of milk mononuclear cells and cytokines during the involution of the mammary gland.

METHOD OF STUDY: Bioassays were used to determine the levels of transforming growth factor beta (TGF‐β) and interleukin‐2 (IL‐2) and their effects on milk mononuclear cells (MMCs). RESULTS: MMCs collected from involuting glands were less responsive to Con A stimulation than peripheral blood mononuclear cells (PBMCs), and purified huTGF‐β1 inhibited the activation of both MMCs and PBMCs by Con A. Furthermore, secretions collected over a period of approximately 4 weeks into the involution period contained high levels of active TGF‐β and extremely high levels of latent TGF‐β. MMCs stimulated with Con A produced higher levels of IL‐2 than did the PBMCs of the same animals, and bovine milk TGF‐β and huTGF‐β1 significantly inhibited the IL‐2 production by MMCs. Mammary secretions of involuting glands did not contain any detectable levels of IL‐2, and they inhibited the growth of the IL‐2‐dependent bovine lymphoblastoid T cell line (BLTC), even in the presence of 10 U/ml of rBo IL‐2.

CONCLUSIONS: The findings suggest that TGF‐β is a major immunoregulatory factor in the involuting mammary gland.
Membrane Phenotype and Expression of Perforin and Serine Esterases by CD3− Peripheral Blood and Decidual Granular Lymphocyte‐Derived Clones
Authors: Gudelj L. , Christmas S.E. , Laskarin G. , Johnson P.M. , Podack E.R. , Rukavina D.
Published: September 2011

PROBLEM: Human first‐trimester pregnancy decidua were found to contain large numbers of perforin (P)‐containing cells, which varied in their membrane antigen phenotype. In this study results obtained by analyzing CD3 clones derived from human early pregnancy decidua and peripheral blood are reported.

METHOD OF STUDY: Decidual tissue was obtained from vaginal termination of first trimester normal human pregnancies. CD3− clones were generated by limiting dilution cloning after the depletion of CD3+ lymphocytes. The cell membrane phenotype was determined by (low cytometry. Perforin was detected by fluorescence‐activated cell sorter (FACS) analysis of permeabilised cells. Serine esterases (SE) were identified by histochemical staining for BLT‐esterase.

RESULTS: Cloned decidual cell populations retained the overall antigenic phenotype of freshly isolated decidual natural killer (NK)‐like cells. AU CD3 clones, either derived from decidua or from peripheral blood contained perforin. Serine esterases were present in every decidual clone analyzed.

CONCLUSIONS: Limiting dilution cloning allows the clear‐cut analysis of homogenous subsets of decidua‐derived NK‐like clones. The presence of large amounts of perforin in all of the CD3− clones underlines the extensive transcription of the perforin gene by NK‐like lymphocytes.
Down‐Regulated Expression of Perforin‐Positive/CD16+ Cells in the Peripheral Blood Lymphocytes in the First Trimester of Pregnancy and Up‐Regulation at the End of Pregnancy
Authors: Rukavina Daniel , Podack Eckhard R. , Rubesa Gordana , Spanjol‐Pandelo Suzana , Randic Ljiljana
Published: September 2011

PROBLEM: Immunophenotypic profiles of perforin (P)‐positive peripheral blood lymphocytes in the first trimester and at the term of human pregnancy were analyzed.

METHOD OF STUDY: Perforin expression in peripheral blood lymphocyte subsets was measured by simultaneous detection of P (intracellular antigen) and cell‐surface antigens (CD3, CD4, CD8, CD16, and CD56) by flow cytometry in nonpregnant (NP) and pregnant women in the first trimester (FTP) and at the time of parturition (TP).

RESULTS: The percentage of total P+ cells in peripheral blood compared to nonpregnant women was slightly lower in the FTP but significantly higher at TP. Perforin‐positive cells were significantly elevated in T lymphocyte subsets (CD3+P+, CD4+P\ CD8+P+) in both the FTP and TP groups, as was the percentage of CD56+P+ cells. Profound changes in the CD16+ subpopulation were found in the FTP group compared to both the NP and TP groups (a drastic decrease of CD16+P+ cells; CD16+ cells among P+ cells; P+ cells among CD16+ cells). A considerable part of CD3+ cells in both the FTP and TP groups are CD3+CD56+P+. The average fluorescence intensity (AFI) for P (a measure of P content per cell) was significantly decreased in FTP and increased in TP groups.

CONCLUSIONS: The CD16 molecule is FcγRIIIA which is the only Fc receptor responsible for antibody dependent cell‐cytotoxicity (ADCC) of NK and T‐cells. In the first‐trimester human pregnancy this mechanism is severely down‐regulated compared to both the NP and TP groups.
Prevalence and Biological Effects of Anti‐Trophoblast and Anti‐Endothelial Cell Antibodies in Patients with Recurrent Spontaneous Abortions
Authors: Tedesco F. , Nardon E. , Pausa M. , Bulla R. , Narchi G. , Guaschino S. , Livi C. , Meroni PL.
Published: September 2011

PROBLEM: Trophoblasts and endothelial cells represent a potential target for antibodies in women with recurrent spontaneous abortions. These antibodies have been shown to be associated with anti‐phospholipid antibodies. Are they also present in women with unexplained pregnancy losses in the absence of anti‐phospholipid antibodies?

METHOD OF STUDY: The anti‐trophoblast antibodies were tested by an immunofluorescence assay on cells purified from pooled first‐trimester placentae, whereas the anti‐endothelial cell antibodies were measured by enzyme‐linked immunoadsorbent assay (ELISA) on cells isolated from the umbilical vein and were cultured to confluence. The cytotoxicity of trophoblasts was evaluated in a homologous system. The expression of adhesion molecules on endothelial cells was quantitated by ELISA using specific monoclonal antibodies, and the expression of tissue factor was quantitated by a chromogenic assay measuring the formation of factor Xa.

RESULTS AND CONCLUSIONS: Complement‐fixing antibodies to trophoblast represent a better marker to discriminate patients with recurrent spontaneous abortions from controls and are cytotoxic for the target cells. Anti‐endothelial antibodies are also present in these patients and exhibit pro‐inflammatory and pro‐coagulant activities.
Antisperm Antibody Detection: 1. Methods and Standard Protocol
Authors: Dondero Franco , Gandini Loredana , Lombardo Francesco , Salacone Pietro , Caponecchia Luisa , Lenzi Andrea
Published: September 2011

PROBLEM: Today, antisperm antibody (ASA) detection is one of the most important steps in the evaluation of male infertility. This practice is generally accepted even though there is still some disagreement about the meaning of antisperm immunity and a good deal of controversy about the test regarded as the most suitable for the detection of antibodies directed against sperm antigens. International workshops have tried to standardize universally accepted protocols. A panel of three or four methods is generally advised to provide a correct and complete screening of patients with antisperm immunity.

METHOD OF STUDY: This paper reports on the results of a serum exchange workshop for the standardization of the ASA detection in the sera carried out under the auspices of the Italian Society of Endocrinology, which was used as reference laboratory. A careful description of the most widely used methods is reported and proposed as a standard protocol also on the basis of the results of the correlation studies carried out by our group is also reported.

CONCLUSIONS: If the proposed methods and procedures will be accepted by the members of the Alps‐Adria Society for Immunology of Reproduction, a Study Group for Sperm Antibody Testing, could start its activity having the goal of reaching a consensus on methodology and carrying out a European serum exchange workshop.
Characterization of Antibodies Generated Against a Conserved Portion of Oviductal Glycoprotein (OGP) and Endogenous Hamster OGP and Their Ability to Decrease Sperm Binding to the Zona Pellucida In Vitro
Authors: Schmidt A. , Mavrogianis P.A. , O'Day‐Bowman M.B. , Jaffe M.C. , Verhage H.G.
Published: September 2011

PROBLEM: The effect of antibodies generated against hamster oviductal glycoprotein (OGP) on sperm binding to the zona pellucida (ZP) was evaluated.

METHOD OF STUDY: Antibodies against a 17‐amino‐acid sequence of the OGP core protein (amino acids 52–68) and the denatured hamster OGP protein were generated, characterized, and tested in an in vitro sperm binding assay.

RESULTS: Sperm binding was significantly decreased (P < 0.05) when oviductal oocytes were incubated for 2 hr with 4 or 8 mg/ml of immune IgG of both antibodies when compared with normal rabbit IgG. A fluorescence assay showed binding of both antibodies to the endogenous OGP associated with the ZP of ovulated hamster oocytes. CONCLUSIONS: These results suggest that OGP may be a potential immunocontraceptive target because both antibodies significantly decreased sperm binding to the ZP of oviductal oocytes. Immunocontraception may be accomplished by attempting to generate active immunity to a recombinant OGP, to the region selected in this study (amino acids 52–68) or to some other region of the core protein.
The Presence of a C1‐Inhibitor‐Like Molecule (C1‐INH‐L) On Human Sperm: Its Involvement in Sperm Motility
Authors: Jiang Haixiang , Pillai Subbi , Ruby Eric , Howard Leslie , Butler William J. , Zhang Huamei
Published: September 2011

PROBLEM: An 88–92‐kDa C1‐inhibitor‐like molecule (C1‐INH‐L) was previously identified to elicit cytotoxic sperm antibody response in infertile men and women. Here, we document that it is present on the human sperm surface and could be detected by an enzyme‐labeled immunoglobulin G (IgG) fraction of anti‐human C1‐INH antibody. METHOD OF STUDY: Western blot analysis, enzyme‐lined immunoadsorbent assay (ELISA) and computerized sperm motion analysis.

RESULTS: The existence of C1‐INH‐L on the sperm surface is calcium independent. Phosphatidylinositol‐specific phospholipase C (PIPLC), EDTA, and acid (pH 3.0) could not remove the C1‐INH‐L from sperm, but trypsin did. Activated C1s was able to bind to the sperm surface. Immunofluorescence studies localized the protein to the head and midpiece of the sperm membrane. The C1‐INH‐L exists on both uncapacitated and capacitated sperm surfaces, which suggests that this protein is a sperm‐surface protein. The heat‐treated (56°C, 30 min) IgG fraction of anti‐C1‐INH greatly reduced the percentage of motile spermatozoa and the progressive and path velocities in the absence of complement. CONCLUSION: Our data suggest that C1‐INH is a sperm membrane‐anchored protein that may have complement and sperm motility regulatory function.
Lymphokine‐Activated Killer (LAK)‐Mediated Lysis of Sequentially Isolated Ovarian Cancer Cell Lines
Authors: Yashar Catheryn M. , Taylor Duglas D. , Gibb Rndall K. , Gerçel‐Taylor çiçk
Published: September 2011

PROBLEM: Understanding immunologic eradication of cancer is hampered by failure to explore tumor evolution. Cell surface molecules may alter with therapy and disease progression. These alterations can translate into variable susceptibility to immune‐mediated cell lysis.

METHOD OF STUDY: Cell lines from a patient with ovarian carcinoma isolated at surgical debulking (UL‐3A), during chemotherapy (UL‐3B), and after progression (UL‐3C) were used to study changes in cell lysis by natural killer (NK) and lymphokine‐activated killer (LAK) cells. The role of adhesion molecules ICAM‐1, LFA‐3, and glycoproteins in the demonstrated differential killing was also examined.

RESULTS: An inverse relationship between attachment and lysis was demonstrated. UL‐3C, the most sensitive to lysis (50%), attached the least lymphocytes (40%), whereas UL‐3A, the least sensitive (33%), attached the most lymphocytes (71%). A correlation with ICAM‐1 and LFA‐3 expression was not demonstrated.

CONCLUSION: Ovarian cancer cells evolve throughout the disease course, and this may manifest as differential sensitivity to immune‐mediated cell lysis.