bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevention of Pregnancy‐Induced Hypertension in Twins by Early Administration of Low‐Dose Aspirin: A Preliminary Report
Authors: Caspi Eliahu , Raziel Arieh , Sherman Dan , Arieli Shlomo , Bukovski Ian , Weinraub Zvi
Published: May 2013

PROBLEM: Effectiveness of early administered low‐dose aspirin in prevention of pregnancy‐induced hypertension (PIH) and fetal growth retardation in twin pregnancies was investigated in a randomized placebo controlled, double‐blind trial in 47 twin pregnancies.

METHOD: Twenty‐four women received 100 mg of aspirin daily from mean gestational age of 17.7 wk, and 23 women ingested placebo from a mean gestational age of 18 wk until delivery. The placebo and aspirin group were similar in age, weight gain, zygosity, gravidity, parity, and obstetrical antecedents. Treatment lasted for a mean period of 16.8 wk and 18.3 wk in the placebo and aspirin groups, respectively. The mean gestational age at birth was 35.0 wk and 36.4 wk in the placebo and aspirin groups, respectively.

RESULTS: PIH was noted in six women (26%) in the placebo group, but in only one woman (4%) in the aspirin treated patients (P<.05). The mean combined fetal weights of both twins, and the mean weight of the second twin at delivery were significantly higher in the aspirin treated mothers than in the placebo treated gravidas (mean difference of 781 g, P<.02 and mean difference of 488 g, P < .005, respectively). Intrauterine growth retardation (< 10th percentile) concerned 11 (24%) and six (13%) fetuses in the placebo and aspirin groups, respectively. No adverse effects of treatment to either the mothers or the infants were noted.

CONCLUSION: Low‐dose aspirin reduces the incidence of PIH and has a beneficial effect on fetal growth in twin pregnancies. Additional clinical trials are needed in order to define and select subgroups of twins where aspirin treatment is recommended.
Antisperm Antibodies (ASAs) in Infertile Males: Subclass Distribution of IgA Antibodies and the Effect of an IgA1 Protease on Sperm‐Bound Antibodies
Authors: Kutteh William H. , Kilian Mogens , Ermel Leann D. , Byrd E. William , Mestecky Jiri
Published: May 2013

PROBLEM: (1) To determine the IgA subclass distribution of antibodies in the serum and on the sperm of infertile male patients. (2) To determine the effect of an IgAl protease on the binding of IgA antisperm antibodies (ASA).

METHOD: Fifteen infertile males with ASA in serum (10) or on sperm (5) were recruited for this study. Duration of infertility was at least one year. Monoclonal antibodies to human IgAl and IgA2 were conjugated to immunobeads. The distribution of IgAl and IgA2 subclass ASA was determined for positive serum and sperm samples. The effects of an IgAl protease (isolated from Neisseria meningitidis strain HF13) on sperm‐bound antibodies was evaluated by immunobead binding.

RESULTS: In serum, the IgAl subclass ASA was predominate (91%) when compared with IgA2 (9%) subclass. Direct sperm‐bound antibodies displayed a distribution more characteristic of the secretory immune system with IgAl accounting for 63% and IgA2 accounting for 37% of the total IgA ASA. Enzyme treatment dramatically reduced the amount of serum IgA antibodies bound to sperm (P < 0.05). Similarly, a significant reduction in direct sperm‐bound antibodies was observed after enzymatic treatment with no loss in sperm motility.

CONCLUSIONS: (1) Although IgAl direct sperm‐bound antibodies were dominant, when compared with serum there was a higher proportion of IgA2 subclass, which suggests a local production of IgA. (2) Specific IgAl protease treatment is capable of reducing the amount of immunobead‐detectable IgA on sperm. Hamster oocyte sperm‐penetration assays are ongoing to determine if this treatment might improve sperm penetration rates.
Octreotide Effect on Ovarian Morphology in Insulin‐Resistant PCOS Patients Following Six‐Month Decapeptyl Treatment
Authors: Goni Marie‐Helene , Markussis Viron , Tolis George
Published: May 2013

PROBLEM: Regulation of ovarian folliculogenesis involves bidirectional communication between the immune and endocrine systems. Somatostatin analogues have been reported to acutely suppress elevated androgens in polycystic ovary syndrome (PCOS). The aim of our study was to analyze the morphologic and hormonal‐metabolic response to octreotide therapy for one month in insulin‐resistant PCOS patients in whom luteinizing hormone (LH) effect had formerly been separated by a six‐month GnRH‐agonist (GnRH‐a) course.

METHOD: Fifteen PCOS patients were studied two months after completing a six‐month GnRH‐a (decapeptyl 3.75 mg monthly injection) course. Seven of the patients (group A), who were insulin‐resistant and gave hyperinsulinemic response to a glucose challenge, received a 50‐μg subcutaneous injection of octreotide twice a day for one month. The nonhyperinsulinemic patients (group B) received placebo injections. Hormonal measurements, oral glucose tolerance test (OGTT), and transvaginal ovarian ultrasound were performed before and toward the end of the treatment period.

RESULTS: After octreotide ovarian volume dropped significantly in group A (× ± SD) (19.2 + 5.1 versus 14.7 ± 5.5 cc, P = .02). LH levels increased (3.25 ± 1.22 versus 5.95 ± 4.34 mu/ml, P = .05) as did E2 levels (38.0 ± 11.4 versus 55.1 ± 12.7 pg/ml, P = .005). There was no change in follicle‐stimulating hormone, 17‐hydroxy‐progesterone, free testosterone, or androstenedione levels. Insulin secretion during OGTT dropped significantly (555 ± 294 versus 68 ± 29 μu/ml/hr, P = .002). Glucose tolerance was not affected. In contrast, the placebo‐treated group B patients showed an increase in ovarian volume (10.9 ± 3.5 versus 14.8 ± 3.3 cc, P = .001) while their gonadotropin and steroid profile relapsed, similarly to our patients receiving octreotide.

CONCLUSIONS: Octreotide has an adjunctive beneficial effect to GnRH‐a on ovarian morphology although, at the dose used, there was no suppression of gonadotropin or ovarian steroid levels. The changes in ovarian morphology are probably mediated through suppression of insulin levels and/or other ovarian growth factors.
Separation of a 170‐K Delayed‐Implantation‐Associated Protein With Inhibitory Activity on Trophoblast Outgrowth and RNA Synthesis by Mouse Embryo
Authors: Katagiri Seiji , Yuen Basil Ho , Moon Young S. , Takahashi Yoshyuki , Kanagawa Hiroshi
Published: May 2013

PROBLEM: To screen the uterine protein responsible for embryonic dormancy associated with delayed implantation.

METHOD: Uterine protein extracts and sera from mice in which delayed implantation had been induced and those from pregnant mice were separated by three steps of chromatography and SDS‐PAGE by monitoring an inhibitory activity on trophoblast outgrowth. The presence of the separated protein in the uterine luminal fluid was assessed. Effect of the protein on cell proliferation and RNA synthesis by blastocysts were assessed.

RESULTS: A 170‐K protein was found in the uterine tissue as well as uterine luminal fluid associated with delayed implantation. The 170‐K protein suppressed RNA synthesis by approximately 50% and cell proliferation in blastocysts.

CONCLUSION: A 170‐K protein is secreted into the uterine lumen during delayed implantation period. The ability of 170‐K protein to suppress RNA synthesis and cell proliferation may play a role in regulation of embryonic dormancy associated with delayed implantation.
Lactation Stage‐Dependent Changes in Levels of Tumor Necrosis Factor/Cachectin in Milk
Authors: Rewinski M.J. , Yang T.J.
Published: May 2013

PROBLEM: Determination of lactation stage‐dependent changes in levels of tumor neurosis factor (TNF) in milk.

METHOD: Bioassay and immunoblocking assay were used to identify and assay tumor necrosis factor (TNF; mostly TNFα) in bovine milk at different stages of lactation.

RESULTS: TNFα levels in milk started to increase steadily after the onset of drying‐off (weaning/involution), peaked at 4 to 6 wk prior to parturition and precipitously decreased to undetectable levels at parturition (colostrum). Thereafter, TNFα reappeared and maintained midlevel concentration in the mature (normal) milk throughout the rest of the lactation cycle. Analysis of cells in mammary secretions by flow cytometry revealed that elevated TNFα levels coincided with an increase in macrophages in the secretion from the dry period.

CONCLUSIONS: These lactation stage‐dependent changes in TNFα levels reflect differential effects that TNFα have on involution and prepartum remodeling of the mammary gland of the dam and on gastrointestinal development and immunoregulatory function of the suckling.
Anti‐Sperm Antibodies in Female Mice: Responses Following Intrauterine Immunization
Authors: Stern Judy E. , Nelson Tina S. , Gibson Sarah H. , Colby Ester
Published: May 2013

PROBLEM: The goal of this study was to measure anti‐sperm antibodies in serum following intraluminal uterine immunization and to compare this with the formation of antibodies following intraperitoneal (i.p.) or anal immunization.

METHOD: Mice were immunized with human sperm, and antibody was measured using an indirect immunobead assay.

RESULTS: High levels of anti‐sperm antibodies could be measured following intrauterine immunization and i.p. immunization. Anal immunization resulted in lower levels of antibody formation. Antibody formation after intrauterine immunization was less consistent following transcervical insertion than following needle insertion of sperm.

CONCLUSION: The data suggest that intrauterine immunization can result in anti‐sperm antibody formation under some conditions.
Association of Sperm Antibodies With Other Autoantibodies in Infertile Men
Authors: Paschke R. , Bertelsbeck D. Schulze , Tsalimalma K. , Nieschlag E.
Published: May 2013

PROBLEM: In many autoimmune diseases there is an increased incidence of other autoantibodies. However, the incidence of other autoantibodies in patients with seminal sperm antibodies is unknown. The most widely used tests to detect seminal and serum sperm antibodies are the mixed antiglobulin reaction (MAR) and the Tray agglutination test (TAT).

METHOD: We therefore determined the incidence of antinuclear, antimitochondrial, thyroid peroxidase, and thyroglobulin antibodies, and rheumatoid factor in 147 patients investigated with MAR and 157 patients investigated with TAT.

RESULTS: TAT positive patients had a significantly elevated incidence of antinuclear antibodies (χ2 test, P < 0.005) and thyroglobulin antibodies (χ2 test, P < 0.001). Thyroglobulin antibodies were increased in patients with MAR IgG > 40% and also significantly (χ2 test, P < 0.05) increased in MAR IgA positive patients. Furthermore, thyroid peroxidase antibodies were only found in TAT positive patients.

CONCLUSIONS: The consistently increased incidence of thyroid autoantibodies in infertile patients with sperm antibodies may indicate an increased risk for the development of autoimmune thyroid disease. This finding therefore suggests screening of patients with immunologic infertility for autoimmune thyroid disease and a further evaluation of the prognostic and pathophysiologic significance of thyroid autoantibodies in immunologic infertility.
Anti‐ZP3 Antibodies Binding to the Human Zona Pellucida: Effect of Oocyte‐Storage Conditions
Authors: Hinsch Klaus‐Dieter , Hinsch Elvira , Pfisterer Susanne , Schill Wolf‐Bernhard , Gibbons William E. , Hodgen Gary D. , Oehninger Sergio
Published: May 2013

PROBLEM: The zona pellucida protein 3 (ZP3) is a zona pellucida (ZP) glycoprotein crucially involved in fertilization. ZP3 plays a major role in sperm binding and induction of the acrosome reaction. In different species, ZP3 proteins differ in their primary structure as derived from cDNA clones. The hemizona assay (HZA) is a bioassay that evaluates binding of human sperm to human ZP and is highly predictive of fertilization outcome under in vitro conditions.

METHOD: In these studies, we used antisera generated against synthetic ZP3 peptides to compare antibody binding patterns to ZP with sperm‐ZP binding capacity under different HZA conditions.

RESULTS: Analysis of antibody binding to hemizonae derived from metaphase II human oocytes that were used either after refrigeration at 4°C or stored in a hyperosmotic salt solution revealed a strong reaction with human ZP3. However, treatment of human oocytes using a protocol to freeze embryos with the addition of 1,2 propanediol drastically reduced binding of ZP3 antibodies to the hemizonae. Nevertheless, no significant difference of sperm binding occurred under HZA conditions when oocytes were refrigerated, salt‐stored, or frozen with 1,2 propanediol.

CONCLUSIONS: Our results indicate that the ZP3 protein backbone might be altered by 1,2 propanediol‐treatment while the glycoprotein‐receptor remains intact. We conclude that antisera against ZP3 peptides can be used as markers for the ZP3 protein backbone in human oocytes and might be useful tools for the evaluation of ZP3 protein integrity.
Myometrial Expression of mRNA Encoding Epidermal Growth Factor Receptor (EGFR) Throughout the Menstrual Cycle
Authors: Heiner James S. , Cai L. , Ding H. , Rutgers J.K.L.
Published: May 2013

PROBLEM: To investigate the quantitative changes in expression of EGFR mRNA in the myometrium throughout the menstrual cycle.

METHOD: Myometrium was collected at hysterectomy from 27 women with a history of regular cycles. Total RNA (20 μg) was isolated and analyzed by Northern blot using a human EGF‐R specific 32P‐labeled cDNA probe. The hybridization signals were quantified by densitometry, standardized, and reported in densitometry signal units (DSU). Endometrial specimens from the same uteri were simultaneously evaluated for histologic dating of menstrual cycle day. EGFR gene expression in endometrium was previously reported. Statistical significance of the differences in myometrial gene expression between menstrual phases was evaluated by student's t test.

RESULTS: EGFR mRNA was expressed in all myometrial tissues tested. Levels were higher in the late proliferative phase than in all other phases (P < .05). Women over 40 years old had lower proliferative phase expression than younger women.

CONCLUSION: This data suggests that myometrial EGFR mRNA expression varies in association with the histologic phases of the normal menstrual cycle, and may be affected by aging, even when cycles occur at regular intervals.
Gestational Age Correlates With Immunosuppressive Properties of Hydatidiform Mole Pregnancies
Authors: Bennett William A. , Long Cecil A. , Mcgehee Ramon P. , Cowan Bryan D.
Published: May 2013

PROBLEM: Soluble trophoblast extracts (HME) from some human hydatidiform mole pregnancies suppress IL‐2‐dependent T‐cell proliferation, while others express no immunosuppressive bioactivity. This study was designed to determine if suppression by HME was correlated with gestational age, uterine size, or hCG secretion.

METHOD: Soluble extracts were prepared from nine hydatidiform mole trophoblast samples and screened for immunosuppressive activity using a murine cytotoxic T‐cell proliferation assay (CTLL‐2). Gestational ages were determined from last menstrual cycle and uterine size was estimated at the time of surgery. Serum samples were collected prior to uterine evacuation and were assayed for human chorionic gonadotropin (hCG).

RESULTS: Four of nine HME samples significantly (P < 0.05) suppressed CTLL2 proliferation, while five exhibited no suppressive activity. A strong positive correlation (r = 0.639) was noted for the relationship between gestational age of the molar pregnancies and interleukin‐2(IL‐2)‐stimulated CTLL2 proliferation (expressed as % of control) in the presence of HME (500 μg/mL). This indicates that HME suppression of CTLL2 proliferation is highest in early gestation and then declines with increasing gestational age. A similar correlation was observed between estimated uterine size at surgery and CTLL2 proliferation with added HME, although the association was not as strong (r = 0.359). No association was noted between hCG levels and CTLL2 proliferative responses (r = −0.091).

CONCLUSIONS: The results of this study suggest that production of immunosuppressive factors by hydatidiform mole trophoblast is developmentally regulated, and decreases with advancing gestation.
Nuclear Regulation of HLA Class I Genes in Human Trophoblasts
Authors: Chiang Mimi H. , Main Elliott K.
Published: May 2013

PROBLEM: Human trophoblast expression of class I human leukocyte antigen (HLA) genes is unique in that there is no classical gene expression, but nonclassical HLA‐G is expressed and only by cytotrophoblast cells. This differential expression of classical versus nonclassical class I genes suggests tissue specific regulation. Recently, a negative regulatory element (NRE), 180 bp 5′ to transcription initiation was identified in a murine embryonal carcinoma cell line that markedly inhibited class I gene expression (Flanagan et al., Proc Natl Acad Sci USA 1991; 83:3145–3149).

METHOD: Here we analyzed the human HLA‐A2 gene for a putative NRE sequence and determined whether such a sequence is capable of binding to factors present in a variety of class I‐null cell lines.

RESULTS: Sequence analysis revealed that the NRE for human HLA‐A2 is identical to that for mouse H‐2Ld. Using gel shift assays with nuclear extracts (NE) from a variety of cell types, we demonstrated specific binding to the HLA‐A2 NRE sequence. The choriocarcinoma cell lines JEG and BeWo and the F9 cells (all negative for classical gene expression) contained this DNA binding factor(s). This binding factor was not present in NE from lymphocytes or a variety of other cell lines that were positive for classical gene expression.

CONCLUSION: Human trophoblasts appear to have a tissue specific nuclear binding factor that may down regulate classical class I expression upon binding to the NRE sequence. The HLA‐G gene does not have this NRE region thus enabling its expression by these cells.
Characterization of Lymphocyte Subpopulations and T Cell Activation In Endometriosis
Authors: Witz Craig A. , Montoya Iris A. , Dey Tammy D. , Schenken Robert S.
Published: May 2013

PROBLEM: Numerous studies have characterized the lymphocyte subpopulations in normal eutopic endometrium and suggested a role for the cytokine secretory products of these lymphocytes in regulating endometrial cell proliferation and differentiation. Recent studies have shown that ectopic endometrium contains a greater concentration of scattered stromal lymphocytes than does eutopic endometrium. However, the lymphocyte subpopulations and their activation status have not been characterized in ectopic endometrium.

METHODS: We performed immunohistochemical studies on serial sections of proliferative and secretory phase eutopic endometrium and ectopic endometrium obtained during the proliferative phase using monoclonal antibodies to CD4 (T helper‐inducer cells), CD8 (T cytolytic‐suppressor cells), CD22 (B‐cells), CD56 (natural killer cells), and VLA‐1 (T‐cell activation marker).

RESULTS: Ectopic endometrium contained significantly more scattered stromal CD4, CD8, and activated T cells than did proliferative and secretory eutopic endometrium. There were more activated T‐cells in proliferative than in secretory eutopic endometrium. Ectopic endometrium contained significantly fewer NK cells than proliferative and secretory endometrium.

CONCLUSIONS: These results demonstrate that (1) the increased lymphocyte population in ectopic endometrium is due to increased numbers of CD4 and CD8 cells, and (2) a greater number of activated T cells are present in ectopic endometrium as compared to eutopic endometrium. Increased concentration of stromal T cells and enhanced VLA‐1 expression in ectopic endometrium suggest that cytokine products of the activated T‐cells may be involved in regulating cellular processes of endometriosis tissue.
Interferon‐Gamma (IFN‐γ) and Interleukin‐6 (IL‐6) in Peritoneal Fluid and Macrophage‐Conditioned Media of Women With Endometriosis
Authors: Keenan Jeffrey A. , Chen Thomas T. , Chadwell Nancy L. , Torry Donald S. , Caudle Michael R.
Published: May 2013

PROBLEM: The presence of the various cytokines in human peritoneal fluid has been incompletely evaluated. Changes in cytokine levels may be related to activation of peritoneal macrophages, development of endometriosis, and infertility. This study assesses peritoneal fluid levels of interferon gamma (IFN‐γ) and interleukin‐6 (IL‐6), and peritoneal macrophage production of IL‐6, in women with and without endometriosis.

METHOD: Peritoneal fluid was obtained from 62 women at the time of diagnostic or operative laparoscopic surgery for benign gynecologic disease. Peritoneal macrophages were isolated, cultured for 24 h, and the culture media collected. IFN‐γ and IL‐6 levels in peritoneal fluid samples and macrophage conditioned media were determined by commercial ELISA.

RESULTS: IL‐6 was significantly higher in the macrophage conditioned media of women with endometriosis as compared with controls. IL‐6 levels were fourfold higher in early stage endometriosis (P < 0.05) and eightfold higher in advanced endometriosis. There were no significant differences between groups in the peritoneal fluid levels of IL‐6 or IFN‐γ.

CONCLUSIONS: Peritoneal macrophage IL‐6 secretion is increased in women with endometriosis, and appears to correlate with disease stage. IFN‐γ does not appear to be responsible for the activation of macrophages in women with endometriosis.
Protein Phosphorylation Pattern and Role of Products of c‐erbB‐1 and c‐abl Proto‐oncogenes in Murine Preimplantation Embryonic Development
Authors: Ahmad Khaliq , Naz Rajesh K.
Published: May 2013

PROBLEM: To investigate the protein phosphorylation pattern and role of products of c‐erbB‐1 and c‐abl proto‐oncogenes with known tyrosine kinase activity in preimplantation embryonic development in mice.

METHOD: The protein phosphorylation pattern was studied by in vitro 32P metabolic labeling of murine ova/embryos as well as by in vitro kinase assay performed directly on various ova/embryos extracts. The role of products of c‐erbB‐1 (170 kDa, receptor for epidermal growth factor [EGF]) and c‐abl proto‐oncogenes (150 kDa) was examined by in vitro culturing murine embryos in the presence of monoclonal antibodies to respective protein products and by co‐culturing with EGF, the ligand for EGF receptor (EGF‐R).

RESULTS: In vitro metabolic labeling of murine ova/embryos showed 32P incorporation into at least two protein bands of murine ova (Mr 81 and 36 kDa), six protein bands of two‐cell (Mr 81, 36; and 97, 52, 22 and 19 kDa, respectively), six protein bands of morula (Mr 81, 36; 97, 22, and 19; and 33 kDa, respectively), and eight protein bands of blastocyst (81, 36; 97, 22, 19; and 115, 58, and 15 kDa, respectively), stage embryos; there were some specific bands in each stage. Prolonged labeling from 2 to 4 h not only resulted in a relative increase in 32P incorporation into these proteins but also revealed additional bands in morula (Mr 133 and 115 kD) and blastocyst (Mr 49, 33, and 31 kD) stage embryos. In vitro kinase assays performed directly on various ova/embryos extracts revealed at least three phosphoproteins (Mr 58, 36 and 33, respectively) that were common to ova, two‐cell, morula, and early/late blastocyst stage embryos. Additionally, three protein bands each in murine ova and two‐cell embryos (Mr 108, 81, 73 kDa, respectively), and four protein bands of late blastocyst (Mr 108, 73; 133 and 18 kDa, respectively) stage embryos were also revealed. Culture of two‐cell embryos in the presence of EGF, the ligand for EGF‐receptor, resulted in a concentration dependent increase (P< .001) in the number of cells per blastocyst. Monoclonal antibody to c‐erbB‐1 170 kDa protein (receptor for EGF) did not affect development of in vitro cultured murine embryos from two‐cell to morula, but significantly (P<.001) inhibited the in vitro development of morula to late blastocyst stage. Monoclonal antibody to c‐abl protein inhibited the development of murine embryos from two‐cell to morula (P<.017), as well as, from morula to late blastocyst stage (P<.002 to .01).

CONCLUSIONS: These results suggest that the stage‐specific protein phosphorylation pattern and specific products of c‐erbB‐1 and c‐abl proto‐oncogenes may have a role in preimplantation embryonic development in mice.
Effects of GnRH Antagonist on Lymphocyte Subpopulations in Primary and Secondary Lymphoid Tissues of Female Mice
Authors: Rao L. V. , Cleveland R. P. , Kimmel R.J. , Ataya K. M.
Published: May 2013

PROBLEM: GnRH analogs are playing an increasing role in the treatment of many clinical disorders. Recent studies have indicated that GnRH agonists suppress immune function in mice in vivo. The present study investigated the effects of GnRH antagonist on functional lymphocyte subsets of mice in vivo.

METHOD: Three‐ and 10‐wk old female mice received 10 μg of Nal‐Glu daily for 15 and 30 days; changes in the immunophenotypic expression of lymphocytes from thymus, bone marrow, spleen and blood were analyzed by flow cytometry.

RESULTS: The administration of GnRH antagonist to pre‐ and postpubertal female mice induced slight increases in lymphocyte subpopulations in primary and secondary lymphoid tissues. These effects are opposite those obtained with GnRH agonist in our earlier studies in mice.

CONCLUSIONS: Assuming similar effects in humans and rodents, the gonadal steroid suppression achieved by GnRH antagonist treatment has no apparent suppressive effects on the immune system.
Alternative Treatment to Lymphocyte Immunization for Treatment of Recurrent Spontaneous Abortion: Immunotherapy With Intravenous Immunoglobulin for Treatment of Recurrent Pregnancy Loss: American Experience
Authors: Coulam Carolyn B.
Published: May 2013

PROBLEM: Recurrent spontaneous abortion (RSA) is the cause of childlessness in 2–5% of reproducing couples. Immunological mechanisms have been proposed as an etiology in some cases of RSA. Various forms of immunotherapy have been attempted in individuals thought to have an immunologic mechanism associated with RSA. Intravenous immunoglobulin (IVIG) has been used in a pilot study to successfully treat women with RSA.

METHOD: To evaluate the efficacy of IVIG in the prevention of RSA as prospective randomized, placebo‐controlled clinical trial was undertaken. Women experiencing two or more consecutive RSAs receive either IVIG 500 mg/kg/month or placebo (albumin).

RESULTS: To date 92 women have been enrolled in the study and 58 pregnancies have been achieved. The outcome of the 58 pregnancies include 20 deliveries, 9 ongoing pregnancies and 29 losses. Fourteen (49%) of the 29 pregnancy losses were blighted ova (empty gestational sacs) and 15 (51%) were intrauterine fetal deaths (IUFD's) occurring in the first trimester of pregnancy. Of 14 blighted ova, 8 were in women receiving IVIG and 6 were receiving placebo. Fifteen IUFD's occurred: 3 (20%) in women receiving IVIG and 12 (80%) placebo. Of 11 pregnancy losses occurring in women receiving IVIG, 8 (73%) were blighted ova and 3 (27%) were IUFD's. Eighteen losses occurred in women receiving placebo: 6 (33%) were blighted ova and 12 (67%) were IUFD's.

CONCLUSIONS: These preliminary data suggest IVIG may be effective treatment for RSA. Analysis of data from the completed randomized placebo‐controlled trial will test this suggestion.
Predictive Factors in Recurrent Spontaneous Aborters—A Multicenter Study
Authors: CAUCHI MAURICE N. , COULAM CAROLYN B. , COWCHOCK SUSAN , HO G N. , GATENBY PAUL , JOHNSON PETER M. , LUBS MARIE LOUISE E. , MCINTYRE JOHN A. , RAMSDEN GORDON H. , SMITH J. BRUCE , SHARPE KEN
Published: May 2013

PROBLEM: Compare data from several centers relating to success rates in recurrent spontaneous miscarriage and assess the significance of indicators of subsequent pregnancy loss.

METHOD: Data from 777 couples with unexplained recurrent spontaneous abortion from independent studies at seven centers were analyzed using logistic regression analysis. The following covariates were considered: age of patient, number of previous spontaneous abortions, length of previous abortions history, sub‐fertility index (defined as the product of the number of spontaneous abortions and the abortion history), whether a patient was a primary or secondary aborter, and whether a patient had received leukocyte immunotherapy.

RESULTS: There was a highly significant difference between the seven centers in success rates in the subsequent pregnancy and a highly significant association between success rate and each of the following covariates: the number of previous abortions, the length of the previous abortion history and the sub‐fertility index. In particular, for each increase of 10 units in the value of the sub‐fertility index, up to a value of 30, the odds in favor of a successful pregnancy decreased by a factor of 0.6, i.e., 40%. There was, however, little evidence of an association between the success rate in the subsequent pregnancy and age, parity, or immunization with cells from the husband.

CONCLUSIONS: The sub‐fertility index may be a useful measure of likelihood of success in a subsequent pregnancy.
Fertility Among Women With Recurrent Spontaneous Abortions—The Effect of Paternal Cell Immunization Treatment
Authors: COWCHOCK F. SUSAN , SMITH J. BRUCE
Published: May 2013

PROBLEM: The risk of women whose chief complaint is recurrent spontaneous abortions (RSA) for secondary infertility (infecundability) has not been evaluated prospectively. The effect of paternal mononuclear cell immunization on conception rates is unknown.

METHOD: Two hundred women whose chief complaint was RSA were randomly assigned to be immunized with paternal mononuclear cells either before or after (up to 6 postmenstrual weeks) conception. Fertility rates (both conception and live birth) were evaluated for the group immunized before conception and compared to those for the control group, who were not immunized until after conception, using life table and multiple logistic regression analyses.

RESULTS: Prospectively ascertained, age‐related conception rates for nonimmunized RSA controls appeared to be similar to those for general populations. Immunization before pregnancy had no significant effect (power ± 14%) on rates of conception (66% before, 77% after) or time to conceive (median weeks before 19.5, after 27.0). Live birth rates (before 59%, after 63%) were also similar for both groups (P = 0.7).

CONCLUSION: Women whose only prior complaint was RSA were not at high risk for secondary infecundability, and immunization did not alter either conception rates or time to conceive. Postponement of immunization until after conception did not affect live birth rates for women selected for study because they did not have a history of prior infecundability or early repeated miscarriages.
Diagnostic and Prognostic Significance of Anticardiolipin Antibodies in Patients With Recurrent Spontaneous Abortions
Authors: MELK A. , MUELLER‐ECKHARDT G. , POLTEN B. , LATTERMANN A. , HEINE O. , HOFFMANN O.
Published: May 2013

PROBLEM: The role of ACA in unexplained RSA is controversial. In the present study, diagnostic and prognostic aspects were investigated.

METHOD: One hundred five nonpregnant patients with primary, 29 with secondary RSA, and 209 controls were investigated for IgG‐ACA. Follow‐up studies were done during pregnancy in 76 individuals. IgM‐ACA were tested in a subset of patients.

RESULTS: Elevated ACA levels were significantly more frequent in both patient groups (26 and 24%) than in controls (16%). However, there was no correlation of ACA with various parameters including pregnancy outcome. In ACA‐positive patients with successful pregnancy a significant decrease of ACA values during pregnancy was observed, while ACA remained high in aborting patients. IgG‐ and IgM‐ACA correlated well.

CONCLUSIONS: Although the data from nonpregnant RSA patients does not allow diagnostic or prognostic conclusions to be drawn, sequential testing of ACA‐positive individuals provides the possibility to foresee pregnancy outcome.
A Monoclonal Antibody to an Early Pregnancy Factor‐Induced Suppressor Factor (EPF‐S1) Disrupts Implantation in Mice
Authors: ATHANASAS‐PLATSIS STAVROSIA , HOSKIN MEGAN J. , ROLFE BARBARA E. , CAVANAGH ALICE C. , MORTON HALLE
Published: May 2013

PROBLEM: The importance of EPF during pregnancy has been established previously but the importance of the EPF‐induced suppressor factor EPF‐S1 in pregnancy has to date been unaddressed. Investigations were therefore conducted in order to study this.

METHOD: Monoclonal antibodies to EPF‐S1 were produced, and one antibody, designated R2Tγ, was characterized. Mated mice were passively immunized with R2Tγ and the effect on implantation determined.

RESULTS: Characterization of anti‐EPF‐S1 R2Tγ revealed that it cross‐reacted with EPF‐S1 of different MHC restriction but not with EPF or EPF‐S2. When injected into mated mice on days 1 to 4, R2Tγ had no effect on pregnancy but when injections continued to day 5, pregnancy was affected; the number of embryos implanted on day 7 were significantly less than the number of corpora lutea counted, signifying embryonic loss.

CONCLUSION: These studies show that anti‐EPF‐S1 R2Tγ disrupts implantation in mice when injected on days 1 to 5 of pregnancy but not when injected on days 1 to 4, demonstrating that EPF‐S1 exerts its effects around the time of implantation.
In Vivo Immunosuppressive Effects of Recombinant Ovine Interferon‐tau (Trophoblastin): r.oTP (r.oIFN‐τ) Inhibits Local GVH Reaction in Mice (PLN Assay), Prevents Fetal Resorptions, and Favors Embryo Survival and Implantation in the CBA/J × DBA/2 Mice Combination
Authors: ASSAL‐MELIANI AINES , KINSKY RADOSLAV , MARTAL JACQUES , CHAOUAT GÉRARD
Published: May 2013

PROBLEM: Ovine trophoblastin protein, be it natural or recombinant (oTP, r.oTP), a member of the tau interferon family (r.oIFN‐τ), has been shown to possess immunosuppressive properties in vitro. It acts as a cytostatic agent across species. Indeed, it was immunosuppressive when tested on human and murine lymphocytes in a variety of in vitro immune assays, as it is also on syngenic (ovine) lymphocytes.

METHODS: In the present paper, we first verified that this property to act across species also occurred in vivo assays; r.oTP was able to down regulate a local GVH reaction assay (PLN assay) in mice. We then took advantage of these properties of r.oTP to investigate its in vivo effects during murine pregnancy as there is no ovine equivalent of the murine CBA/ J × DBA/2 resorption prone mating combination.

RESULTS: When given in the postimplantation period, r.oTP drastically boosted resorptions in the CBA/J × DBA/2 matings, as did murine recombinant gamma interferon. However, the same r.oTP treatment in the peri‐implantation period resulted in a reduction in resorptions in this spontaneous abortion system.

CONCLUSION: The data suggested that r.oTP might have acted more by favouring implantation and embryo survival than by preventing the resorption process itself. The mechanisms possibly underlying these effects, as well as the putative uses of r.oTP evolving from these data, are discussed.
Characterization of Latent Transforming Growth Factor‐β From Human Seminal Plasma
Authors: NOCERA MARYANN , CHU T. MING
Published: May 2013

PROBLEM: Human seminal plasma is known to exhibit immunosuppressive activity. Transforming growth factor β (TGF‐β) has been identified as an immunosuppressive factor in human seminal plasma. Biologically active TGF‐β represents a family of 25‐kDa homodimeric proteins linked with disulfide bonds. TGF‐β associates with high molecular weight proteins noncovalently to form a type of latency that is biologically inactive. Quantitative distribution of active form of TGF‐β versus inactive latent form of TGF‐β, and mechanism of the TGF‐β activation in human seminal plasma remain to be elucidated.

PURPOSE: To characterize seminal plasma latent form of TGF‐β, including its concentration, and the mechanism underlying the activation of TGF‐β.

METHOD: Gel filtrations on ACA‐34 and Biogel P‐60 were used to fractionate seminal plasma. TGF‐β was measured by enzyme immunoassay using antibodies specific for TGF‐β1 and TGF‐β2, respectively. Radioreceptor assay with recombinant human [125I]‐TGF‐β1 was applied to qualitatively identify TGF‐β1. Kinetic experiments with various pH, temperature and time, along with protease inhibitors, were performed to delineate the activation mechanism of latent TGF‐β.

RESULTS: Human seminal plasma contained both TGF‐β1 and TGF‐β2, predominantly in latent form. The total concentration of TGF‐β1 averaged 238 ng/ml versus an average of 18 ng/ml for TGF‐β2. The in vitro activation or release of TGF‐β1, from latent TGF‐β1 was achieved only at acidic pH of <4.0, and was time and temperature dependent. At pH 3.7 and 37°C, a significant activation of latent TGF‐β1 was achieved after an incubation of only 15 min, reached the maximum at 120 min, and the activated TGF‐β1 remained relatively stable for at least 24 h. The activation was not inhibitable by a series of protease inhibitors examined, alone or in combination (e.g., phenylmethylsulfonyl fluoride, E‐64, pepstatin, leupeptin, ethylenediamine tetraacetic acid). Competitive radioreceptor assay established the functional identity of TGF‐β1 in human seminal plasma with recombinant human TGF‐β1.

CONCLUSION: Human seminal plasma TGF‐β is biologically activated from high molecular weight latent TGF‐β by acid pH. The acidic environment of female lower genital tract could represent an in vivo physiological condition for activation of seminal plasma TGF‐β that may immunologically protect the integrity of sperm.
Leukocyte Subpopulation Changes in Rats With Autotransplanted Endometrium and the Effect of Danazol
Authors: MATSUBAYASHI HIDEHIKO , MAKINO TSUNEHISA , IWASAKI KEN‐ICHI , MARUYAMA TETSUO , OZAWA NOBUAKI , HOSOKAWA TOMOTOSHI , SOMEYA KEN‐ICHI , NOZAWA SHIRO
Published: May 2013

PROBLEM: This study examines immune cell populations in rats with autotransplanted endometrium and determines the effect of danazol on leukocyte subsets.

METHODS: As an experimental model of endometriosis, an autologous endometrial segment was implanted in the rat peritoneum. We used flow cytometry to analyze lymphocyte subsets in the peripheral blood (PB) and peritoneal fluid (PF) of the following groups of rats: no treatment, sham operation, endometrial implantation, endometrial implantation treated with danazol, and normal rats treated with danazol.

RESULTS: The natural killer (NK) cell population was decreased in both the PB and PF of rats with autotransplanted endometrium. Moreover, NK cells increased in a dose‐dependent manner following danazol administration. Surgery itself increased the number of peritoneal macrophages as compared with the untreated group. This elevation was suppressed partially by endometrium‐implantation and was attenuated by subsequent administration of danazol in a dose‐dependent fashion.

CONCLUSIONS: These data suggest that ectopic endometrial cells may release immunosuppressive factors. This is the first documentation that rats with autotransplanted endometrium show the same immunologic changes as humans with endometriosis, and establishes the utility of this model for the study of endometriosis.
Comparative Analysis of the Immunophenotypes of Decidual and Peripheral Blood Large Granular Lymphocytes and T Cells During Early Human Pregnancy
Authors: GEISELHART ANDREAS , DIETL JOHANNES , MARZUSCH KLAUS , RUCK PETER , RUCK MARGARET , HORNY HANS‐PETER , KAISERLING EDWIN , HANDGRETINGER RUPERT
Published: May 2013

PROBLEM: The functional role of the leukocytes in the decidua is not clear. They may regulate the maternal immune response to the fetal allograft. However, the factors controlling maternal and fetal communication have not yet been identified.

METHOD: A comparative analysis of the phenotypes of decidual and peripheral blood large granular lymphocytes (LGLs) and T lymphocytes in early human pregnancy was performed on decidual tissue and blood samples obtained from ten patients at therapeutic abortion.

RESULTS: Whereas most of the decidual LGLs were found to have a CD56bright++ phenotype, most of the peripheral blood NK cells (90%) showed the classical CD56dim+ phenotype, and only a small proportion were CD56bright+ cells. Another striking difference was found in the expression of very late antigen 1 (VLA‐1, CD49a): Almost all the decidual CD56bright++ LGLs, but virtually none of the peripheral blood CD56+ NK cells expressed this antigen. Further differences were found in the expression of CD16, CD44, CD45RA, CD54, and CD57. There were also differences in phenotype between T cells derived from decidual tissue and those derived from peripheral blood. Approximately 31% of the CD3+ decidual T cells expressed VLA‐1, but this antigen was virtually absent on peripheral blood T cells. A further difference was seen in the expression of HLA‐DR. This activation antigen was found on 32 ± 13% of the decidual T cells but only 8 ± 5% of the peripheral blood T cells. Additionally, the proportion of cells expressing CD38 was higher among decidual than peripheral blood T cells.

CONCLUSION: The findings suggest that both decidual LGLs and a subset of decidual T cells are activated and possibly play a role in the control of trophoblast growth and placental development.
Purification and Partial Characterization of an Early Pregnancy Factor‐Induced Suppressor Factor (EPF‐S1)
Authors: ROLFE BARBARA E. , ATHANASAS‐PLATSIS STAVROSIA , HOSKIN MEGAN J. , MORTON HALLE , CAVANAGH ALICE C.
Published: May 2013

PROBLEM: The immunomodulatory properties of early pregnancy factor (EPF) are mediated through induction of at least two lymphokines, designated EPF‐S1 and EPF‐S2 (previously estimated Mr 15,000 and 55,000 respectively). The activity of the former is MHC‐restricted while the latter is restricted to a locus (or loci) outside the MHC. The present study established further criteria by which EPF‐S1 and EPF‐S2 might be distinguished from each other and compared with other suppressor factors. In addition, techniques have been developed to purify EPF‐S1 to homogeneity.

METHOD: Congenic mouse strains were used to map the genetic restriction of EPF‐S2 in the rosette inhibition test and high performance gel permeation chromatography was used to demonstrate that EPF‐S1 induces EPF‐S2 but not vice versa. Further studies then focused on isolation of this first component of the cascade, EPF‐S1, from immune ascites (from growth in athymic mice of the anti‐EPF‐S1‐producing rat‐mouse hybridoma R2Tγ, in which EPF‐S1 is complexed to antibody). Techniques used were acidification followed by application to Sep‐pak C18 cartridges, high performance cation‐exchange chromatography and two reversed‐phase HPLC steps on a C3 column. Purified material was analyzed by SDS‐PAGE and Edman degradation.

RESULTS: Approximately 10 μg EPF‐S1 were isolated from 60 ml ascitic fluid. Homogeneity of the purified material was demonstrated by SDS‐PAGE, where it ran as a single band of approximate Mr 12,000 coincident with biological activity. Attempts at Edman degradation indicate that the molecule is N‐blocked.

CONCLUSION: Definitive primary characterization of EPF‐S1 must await the preparation and isolation of proteolytic fragments of the molecule, but the present studies establish conditions which make such structural analysis possible.
Analysis of Peripheral Blood Lymphocyte Subsets, NK Cells, and Delayed Type Hypersensitivity Skin Test in Patients With Premature Ovarian Failure
Authors: HOEK ANNEMIEKE , VAN KASTEREN YVONNE , DE HAAN‐MEULMAN MEENY , HOOIJKAAS HERBERT , SCHOEMAKER JOOP , DREXHAGE HEMMO A.
Published: May 2013

PROBLEM: Premature ovarian failure (POF) probably belongs to the group of autoimmune endocrinopathies. Cell‐mediated immune parameters were investigated. Sex steroids have a profound effect on the immune system. POF patients and postmenopausal control women (PM) were tested with or without estrogen substitution.

METHOD: A novel FACS analysis system (using double labeling techniques) was used in 30 patients with POF to enumerate the subjects of peripheral blood lymphocytes and NK cells. Eighteen PM women and 30 healthy men and women served as controls. We also tested the delayed type hypersensitivity skin test (DTH) toward Candida in the POF patient group to be informed on their cell‐mediated immune function.

RESULTS: The numbers of blood lymphocytes, CD3+, CD4+ and CD8+ T cells, were not abnormal in POF patients. However, HLA‐DR+ T cells were increased in POF patients and in PM women (P<0.05). These elevated numbers were partially reversible by estrogen substitution. The number of CD19+ cells (B cells) was elevated, whereas CD3−/CD16+/CD56+ cells (NK cells) were decreased in POF patients (P<0.05), irrespective of estrogen substitution. DTH skin tests toward 0.1% Candidin (0.1 ml intradermal injection) were negative in 11 out of 20 tested POF patients, compared to only 2 out of 10 tested controls (P<0.05).

CONCLUSION: POF patients show numerous immune cell abnormalities. These abnormalities were only partially due to estrogen deficiency. We hypothesize that these abnormalities either lead to ovarian autoimmunity or may have direct effects on the ovarian function.
Direct Production of the Fab Fragment Derived From the Sperm Immobilizing Antibody Using Polymerase Chain Reaction and cDNA Expression Vectors
Authors: SAWAI HIDEAKI , YAMASAKI NORIYUKI , SHIGETA MINORU , KOMORI SHINJI , KARASUYAMA HAJIME , KOYAMA KOJI , ISOJIMA SHINZO
Published: May 2013

PROBLEM: Sperm immobilizing antibodies cause infertility mainly through complement dependent sperm immobilization. To analyze any effect of sperm immobilizing antibody on fertilization, we had already established cell lines that secrete IgM monoclonal antibody (MAb H6‐3C4) and IgG monoclonal antibody (MAb EnBCMGS). The latter was a class‐switched recombinant IgG antibody that shares the same variable region as MAb H6‐3C4. The biological effects of the IgG antibody were also reported previously to eliminate sperm immobilizing or sperm agglutinating activities. However, the method of chemical digestion of IgG had some disadvantage to prepare the purified Fab fragment stably and in large quantities. This time we report a unique method to obtain the recombinant Fab fragments (Fab EnBCMGS) using polymerase chain reaction (PCR) and cDNA expression vectors.

METHOD: Two kinds of PCR primers were designed to make a truncated heavy chain (Fd) gene of MAb EnBCMGS. The amplified Fd gene and light chain gene were ligated into cDNA expression vectors and then transfected into mammalian cells.

RESULTS: Expression of the Fd gene and light chain gene were confirmed by Northern blotting. Secretion of the recombinant Fab fragment from mammalian cells was also confirmed by Western blotting. The Fab fragment showed biological activity as is expected by FACS analysis.

CONCLUSION: This method enables the stable production of genuine Fab fragments of IgG in mammalian cells without any chemical treatment that may be time consuming and affect the quality of the Fab fragments.