Search

Objective To ascertain the impact of uterine leiomyomas on pregnancy outcome, and to determine the effectiveness of myomectomy at the time of caesarean delivery. Methods A retrospective study was conducted on pregnant women with uterine leiomyomas. Clinical information including the course of the pregnancy, mode of delivery, pathology findings, and postpartum course were extracted from medical records and analysed for statistical significance. Results There were 50 pregnancies associated with uterine leiomyomas. During pregnancy, the leiomyomas grew >2cm in 46% of cases. Only 6% were affected by symptoms of red degeneration. There were 2% mid trimester inductions of labour, 6% vaginal deliveries, and 92% caesarean sections. Of the 46 caesarean sections, 47% were due to obstetrical factors and 89% of patients underwent myomectomy at the time of caesarean delivery. The mean blood loss from myomectomy at the time of caesarean section was 260ml (200-700ml), and 5% of patients who underwent myomectomy were transfused. Conclusions It is possible to carry a pregnancy successfully to term when the pregnancy is complicated by uterine leiomyomas. When caesarean delivery is needed, myomectomy can be performed at the time of caesarean section routinely without significant complications. [ABSTRACT FROM AUTHOR]

A 45-year-old woman, nulligravida, who underwent saline infusion sonohysterography for infertility evaluation experienced fever and pelvic pain 7 days after the procedure. Transvaginal ultrasonography and computed tomography of the abdomen and pelvis revealed bilateral tuboovarian abscesses (TOA). Because of persistent febrile morbidity despite treatment with broad-spectrum parenteral antibiotics, the TOA were drained under computed tomography guidance. We believe is the first reported case of TOA after saline infusion sonohysterography. [ABSTRACT FROM AUTHOR]

Various studies have emphasized the importance of identifying the optimal Trigger Timing (TT) for the trigger shot in In Vitro Fertilization (IVF), which is crucial for the successful maturation and release of oocytes, especially in minimal ovarian stimulation treatments. Despite its significance for the ultimate success of IVF, determining the precise TT remains a complex challenge for physicians due to the involvement of multiple variables. This study aims to enhance TT by developing a machine learning multi-output model that predicts the expected number of retrieved oocytes, mature oocytes (MII), fertilized oocytes (2 PN), and useable blastocysts within a 48-h window after the trigger shot in minimal stimulation cycles. By utilizing this model, physicians can identify patients with possible early, late, or on-time trigger shots. The study found that approximately 27 % of treatments administered the trigger shot on a suboptimal day, but optimizing the TT using the developed Artificial Intelligence (AI) model can potentially increase useable blastocyst production by 46 %. These findings highlight the potential of predictive models as a supplementary tool for optimizing trigger shot timing and improving IVF outcomes, particularly in minimal ovarian stimulation. The experimental results underwent statistical validation, demonstrating the accuracy and performance of the model. Overall, this study emphasizes the value of AI prediction models in enhancing TT and making the IVF process safer and more efficient., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article. The authors certify that the submission is original work and is not under review at any other publication., (Copyright © 2024. Published by Elsevier Ltd.)

Objective: To assess the cellular and molecular mechanisms of postoperative adhesion development in a rodent model., Design: Prospective randomized controlled study., Setting: Research laboratory., Patients: Thirty sexually mature female Sprague-Dawley rats., Interventions: Cecal abrasion., Main Outcome Measure(s): At 0, 2, 4, 8, 16, 24, 48, 72, 96, 168, 336, and 504 hours after cecal abrasion, one to three rats were sacrificed (n = 26). Four nonabraded rats served as controls. Peritoneal adhesion status was evaluated and tissue was collected for histologic and immunohistochemical investigation., Results: Postoperative tissue attachments were identified as early as 2 hours after cecal abrasion. Significant local edema and vessel congestion appeared within 2 hours, and cellular proliferation was observed at 24 hours; angiogenesis and tissue proliferation remained present at 2 weeks. beta1 integrin was highly expressed early and was thereafter decreased. Cellular fibronectin was not detectable until 1 week after cecal abrasion., Conclusions: Postoperative adhesions are initiated as rapidly as 2 hours after surgical intervention in this rodent model., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: Fetal cells are shed from the regressing chorionic villi and it is possible to retrieve extravillous cytotrophoblast cells by transcervical sampling. The abundance of trophoblast cells in transcervical samples suggests that this non-invasive approach could distinguish between normal and abnormal pregnancies, such as an ectopic pregnancy (EP) and blighted ovum (BO). We aim to identify and quantify fetal trophoblast cells in the cervical canal during the first trimester to assess their usefulness to predict an abnormal pregnancy., Methods: Patients, age 18-45, presenting with a normal intrauterine pregnancy (IUP; n = 37), diagnosis of EP (n = 10) or BO (n = 5) were enrolled for collection of transcervical specimens using a cytobrush and fixative rinse. Non-pregnant, nulliparous women (n = 7) were included as negative controls. Cells were cleared of mucus by acidification, prepared on microscope slides and labeled with a monoclonal antibody recognizing the trophoblast marker, human leukocyte antigen (HLA)-G. HLA-G positive and negative cells were counted to calculate the ratio of trophoblast cells to total cervical cells., Results: Trophoblast cells were observed in 35/37 normal IUP, 6/10 EP and 4/5 BO specimens. The average frequency of HLA-G positive cells in the normal IUP cervical samples was approximately 1 in 2000, which was 4-fold higher than samples from patients with EP or BO (P < 0.001). Receiver operating characteristic analysis showed that EP and BO pregnancies were distinguishable from normal pregnancies with 93% sensitivity, 95% specificity, 97% positive predictive value and 87% negative predictive value., Conclusions: This pilot study presents evidence that trophoblast cells can be reliably obtained and identified among cervical cells in the first trimester by immunohistochemical staining for HLA-G, and suggests for the first time that abnormal pregnancies may be predictable based on the abundance of trophoblast cells in the cervical canal.

Objective: The purpose of this study was to evaluate whether the dynamics of endometrial stripe thickness during gonadotropin-releasing hormone (GnRH) antagonist pituitary downregulation in in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles are related to implantation and pregnancy outcomes., Methods: This retrospective cohort study evaluated 115 conventional IVF/ICSI cycles. All patients underwent ovarian stimulation with gonadotropins and the GnRH antagonist ganirelix acetate. The endometrial stripe was measured transvaginally daily from the day of initial GnRH antagonist administration to the day of the human chorionic gonadotropin (hCG) trigger and then transabdominally on the day of embryo transfer. We created 5 categories (0-4) of endometrial thickness variation, considering significant a daily variation of 1.5 mm. Our aim was to predict whether the endometrial thickness dynamics or morphologic characteristics were related to the duration of ovarian stimulation, duration of ganirelix use, or estradiol levels during ovarian stimulation and whether they would influence implantation and pregnancy rates., Results: No relationship was found between the duration of ovarian stimulation, duration of ganirelix use, and estradiol level (expressed as the area under the curve), and endometrial thickness dynamics or morphologic characteristics. Despite a thinner endometrial thickness in 37% of the cycles on the day of the hCG trigger compared with the beginning of GnRH antagonist stimulation, there was no correlation between endometrial dynamics and pregnancy outcomes. There was, instead, a positive relationship between a trilaminar endometrial morphologic pattern with a positive pregnancy test result, successful implantation, and ongoing pregnancy (P < .05)., Conclusions: Despite a net decrease in thickness in almost 50% of cases, endometrial dynamics did not correlate with pregnancy outcomes. Conversely, a trilaminar endometrial morphologic pattern on the day of embryo transfer was positively related to pregnancy outcomes.

Purpose: To evaluate whether oocyte quality, implantation and pregnancy outcomes in in vitro fertilization (IVF)/intra-cytoplasmic sperm injection (ICSI) are related to the duration of gonadotropin-releasing hormone (GnRH)-antagonist use or the timing of its initiation., Methods: Retrospective cohort study of 178 conventional IVF/ICSI cycles. All patients underwent ovarian stimulation with gonadotropins and GnRH-antagonist for pituitary down-regulation. Spearman correlations and logistic regression were used for statistical analysis., Results: There was no correlation between the duration of use or the timing of initiation of GnRH-antagonist with oocyte quality or implantation and pregnancy outcomes. Oocyte quality was influenced by the peak estradiol. Implantation was influenced by the patient's age. Early pregnancy loss, by the endometrial thickness on human chorionic gonadotropin-day. Ongoing pregnancy was independent from the variables evaluated., Conclusions: GnRH-antagonist duration of use or starting day did not influence oocyte quality, implantation rates, and pregnancy rates. We hypothesize that a follicle stimulating hormone/luteinizing hormone dose increase when antagonist was started, may have had an impact on our findings.

In this retrospective cohort study of 102 ovarian stimulation cycles for IVF/intracytoplasmic sperm injection using GnRH antagonist and gonadotropins, we sought to assess the effect of high E(2) levels on endometrial stripe thickness and its association with pregnancy outcomes and serum E(2) levels. We found no significant correlation between serum E(2) levels (both peak and area under the curve E(2) concentration) and the endometrial thickness. However, there was a statistically significant inverse relationship with early pregnancy loss (31%) if the endometrial thickness was <9.8 mm (sensitivity 71%; specificity 76%).

Purpose: To determine the prevalence of positive test for Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) infections, and their corresponding Rubella status when undergoing workup for infertility., Methods: Retrospective chart review to determine infection status for UU, MH, CT, and NG as determined by cervical swab, as well as the serum Rubella antibody titer., Results: A total of 46 patients of the patients reviewed were positive for UU (20.1%), three patients were positive for MH (1.3%), five patients were positive for CT (2.2%) and one patient was positive for NG (0.4%). Rubella immunity was confirmed in 90.3% of patients., Conclusion: Approximately one quarter of women presenting to an infertility clinic seeking to conceive were found to have a positive test for UU, MH, CT or NG infection. Additionally, almost 10% of the patients were Rubella non-immune at the time of presentation for infertility evaluation.

Objective: To present an overview of the potential etiologies of premature ovarian failure (POF), with an emphasis on the chromosomal abnormalities, gene mutations and autoimmune disorders., Study Design: This article is based on a literature review of articles on POF using PubMed and MEDLINE for the years 1966-2005 using the keywords premature ovarian failure, trying to ascertain the possible mechanisms of POF reported to date., Results: The etiology of POF is still under investigation, even though a wide range of etiologies has been unveiled, encompassing genetic mutations, chromosomal abnormalities, autoimmune disorders, and iatrogenic (irradiation, chemotherapy) and idiopathic causes., Conclusion: In the majority of POF cases, the etiology remains subtle.

Objective: To identify determinants of the serum concentration of hCG levels after triggering of ovulation with exogenous hCG during controlled ovarian stimulation cycles for in vitro fertilization with or without intracytoplasmic sperm injection., Design: Retrospective cohort study., Setting: University Medical Center., Patient(s): One hundred-fifteen women who underwent conventional in vitro fertilization/intracytoplasmic sperm injection cycles from March 2003 to March 2005., Intervention(s): All patients underwent ovarian hyperstimulation with gonadotropins and GnRH-antagonist for pituitary downregulation. Patients were started on oral contraceptives 1 month before the stimulation. Gonadotropins were administered from stimulation day 1 until the day of the hCG trigger, and GnRH-antagonist was added from the day when at least one follicle reached 14 mm in diameter and continued until hCG administration. The hCG was administered in 5,000-IU, 10,000-IU, or 15,000-IU doses on the day of ovulation triggering., Main Outcome Measure(s): We performed a stepwise multiple regression analysis to predict which variable would influence the serum concentration of hCG when measured the day after the administration of exogenous hCG., Result(s): Body mass index (kg/m2) and number of follicles >14 mm were the only determinants of the hCG concentration (cumulative R2 = 0.30; P<.001). Patient age, estradiol peak, number of oocytes retrieved, length of stimulation, and length of GnRH-antagonist administration in days were not associated with serum hCG levels., Conclusion(s): Knowing that the number of larger follicles and the patient's BMI are the major determinants of the hormone's clearance in the body can help in the hCG dose titration during ovarian stimulation.

Embryo implantation and development are critically dependent upon the spatial and temporal regulation of angiogenesis and localized vascular permeability. A key mediator of these effects is the endothelial cell mitogen vascular endothelial growth factor (VEGF). VEGF has been shown to promote endometrial vascular permeability, fetal vasculogenesis and placental, fetal and maternal angiogenesis. However, the mechanism through which this regulation occurs in the placenta is poorly understood. This study was conducted to determine if the pro-angiogenic cytokines, TNF-alpha and TGF-beta1, affect VEGF expression in human first trimester trophoblasts. Culture of a first trimester trophoblast cell line (HTR-8/SVneo), in the presence of either TNF-alpha or TGF-beta1, resulted in the expression of significant levels of VEGF in culture. The trophoblast cell line also showed a time-dependent and a dose-dependent increase in VEGF mRNA levels when cultured in the presence of either TNF-alpha or TGF-beta1. These results suggest that both TNF-alpha and TGF-beta1 may regulate the production of VEGF in early gestational trophoblasts and may therefore serve to modulate placental vascular permeability and angiogenesis that are necessary for embryo implantation and placentation., (Copyright 2000 Harcourt Publishers Ltd.)

Objective: To describe maternal plasma levels of adrenomedullin (AM), a hypotensive and natriuretic peptide, in normal and preeclamptic women at term., Study Design: Maternal plasma AM levels were determined in 13 preeclamptic and 15 normotensive primigravidas by radioimmunoassay. Plasma samples were obtained with the patients in the lateral recumbent position before the administration of any medications., Results: Women with preeclampsia had significantly elevated AM levels when compared with normotensive controls (42.3 +/- 10.5 pg/mL versus 16.9 +/- 3.1 pg/mL, P < .011)., Conclusion: In this pilot study, AM levels were significantly increased at term in preeclamptic women.

Objectives: Altered cytokine expression at the fetoplacental interface may be a potential mechanism for the development of fetal immune dysfunction in children with fetal alcohol syndrome. This study was conducted to determine whether first-trimester trophoblasts respond to ethanol exposure by the induction of specific cytokines., Study Design: HTR-8/SVneo trophoblast cells were cultured in vitro in the presence of either ethanol (0.5% [vol/vol]), lipopolysaccharide (1 microg/mL), or ethanol and lipopolysaccharide. Expression of granulocyte colony-stimulating factor, regulated on activation normal T cell expressed and secreted, and interleukin-6 was examined by Northern analysis and enzyme-linked immunosorbent assay., Results: Culture in the presence of ethanol, lipopolysaccharide, or lipopolysaccharide and ethanol resulted in the increased transcription and secretion of granulocyte colony-stimulating factor, regulated on activation normal T cell expressed and secreted, and interleukin-6 at significantly greater levels (P < .01) than control cultures., Conclusions: Human first-trimester trophoblasts express high levels of cytokines when cultured in the presence of ethanol. Trophoblasts may therefore be an important exogenous source of cytokines for the fetus, and altered cytokine levels during early gestation may have an adverse effect on the development of the fetal immune system.

Objective: Our purpose was to determine whether human choriocarcinoma cells express autocrine motility factor receptor (AMF-R) and to study its function in this tumor system., Study Design: The expression and localization of AMF-R were compared in choriocarcinoma and normal placental trophoblasts using both cell lines and tissue sections. In addition, migratory properties of choriocarcinoma cells and normal placental cells was determined., Results: Using immunofluorescence and immunoperoxidase staining, we have detected the expression of AMF-R in choriocarcinoma cells with receptor clustering on the cell surface, while term placenta cells expressed AMF-R less intensely with no receptor clustering. In choriocarcinoma tissues, AMF-R was strongly expressed in malignant cytotrophoblasts cells while adjacent normal villous trophoblast cells and necrotic regions were weakly or negatively stained. Choriocarcinoma cells responded to AMF-R stimulation with increased cell motility, while term placental cells were unresponsive., Conclusion: Human choriocarcinoma cells express functional cell surface AMF-R in vitro and in choriocarcinoma tissue suggesting that this receptor may play an important role in cancer cell motility.

Problem: Adhesive interaction between trophoblast cells and uterine endometrial basement membrane is one of the critical processes in embryo implantation. This interaction is directly or indirectly regulated by hormones, growth factors, and cytokines. Since tumor necrosis factor-alpha (TNF-alpha) is synthesized by both decidual and trophoblast cells, we hypothesized that TNF-alpha may play a regulatory role in trophoblast cell invasion. To test this hypothesis, we have used in vitro models to determine the effect of TNF-alpha on human trophoblast cell adhesion and motility, two major steps in trophoblast invasion., Methods: The effect of TNF-alpha on the motility of extended-lifespan first trimester trophoblasts (HTR) and JEG-3 choriocarcinoma cells was tested using the phagokinetic track motility assay. An in vitro adhesion assay was used to determine the effect of TNF-alpha on the adhesion of HTR and JEG-3 cells to laminin, a major basement membrane component. In addition, the effect of TNF-alpha on the surface expression of the laminin receptor beta 1 integrin subunit was examined using flow cytometry., Results: HTR or JEG-3 cells strongly adherent to laminin which was not significantly altered by TNF-alpha treatment. We also measured the effect of TNF-alpha on the surface expression of beta 1 integrin on HTR and JEG-3 cells; no difference was observed between control and treatment groups. Interestingly, the motility of both HTR and choriocarcinoma JEG-3 cells was significantly inhibited by TNF-alpha., Conclusions: The role of TNF-alpha in human embryo implantation is currently unknown. Our data demonstrate that TNF-alpha does alter trophoblast cell adhesion to laminin, but significantly inhibits trophoblast cell motility in vitro, suggesting that TNF-alpha may play a regulatory role in trophoblast cell invasion.

Problem: Trophoblast interaction with endometrial extracellular matrix (ECM) is crucial during human embryo implantation and placentation. Entactin, a ubiquitous basement membrane glycoprotein, plays a central role in ECM assembly, cell attachment, and chemotaxis. The present study was conducted to examine the possible role of entactin in promoting human trophoblast adhesion., Methods: Using an extended life span first trimester trophoblast cell line HTR-8/SVneo (HTR) and a cell adhesion assay, we measured the adherence of human first trimester trophoblasts to recombinant entactin and its domains. Also, we used flow cytometry and indirect immunofluorescence to detect the presence of integrins that may be involved in human trophoblast-entactin interaction; these methods were used to analyze HTR cells, as well as tissue sections and freshly isolated human trophoblasts from first trimester and term placenta., Results: We found that first trimester trophoblast cells were highly adherent to entactin and its E and G2 domains but not to G1 or G3 domains. Using indirect immunofluorescence and flow cytometry, we found that both beta 1 and beta 3 integrin subunits were expressed on the surface of HTR trophoblast cells adhering to entactin; in contrast, beta 2 and beta 4 integrin subunits were not detected. In addition, we found that alpha v beta 3 was expressed on freshly isolated villous cytotrophoblasts and cytotrophoblast and syncytiotrophoblasts in tissue sections from term placenta. The beta 3 integrin subunit was expressed in cytotrophoblasts and syncytiotrophoblasts in villi of first trimester placental tissue sections., Conclusion: Recombinant entactin promotes human trophoblast cell adhesion through both its E and G2 domains and these specific adhesive interactions may be mediated by beta 1 and/or beta 3 class integrins.

The objective of this study is to determine the possibility that pre-eclampsia, a disease characterized by altered vascular tone, may result in altered levels of fetal BNP and cGMP, and to determine whether pre-eclampsia alters the maternal-fetal relationship of BNP and cGMP. Paired maternal and umbilical venous plasma levels of BNP and cGMP were determined in 13 pre-eclamptic and 9 normotensive primigravidas in the third trimester. Statistical analysis was performed using multivariate analysis of variance, linear regression, and canonical correlation. Overall, levels of cGMP were lower in pre-eclampsia (P < 0.03). Pre-eclampsia was also associated with an altered maternal-fetal relationship for BNP and cGMP (P < 0.008, P < 0.02, respectively). With pre-eclampsia, the maternal:fetal ratio was reduced for BNP and was increased for cGMP. Because of its role as a second messenger for many vasoactive hormones, we hypothesize that fetal cGMP levels may better reflect overall vascular tone than do individual hormones. Altered BNP and cGMP maternal-fetal homeostasis raises the possibility of maternal-fetal coordination of vascular control.

To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum-free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin-binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell-binding domain (FN-120) and the Arg-Gly-Asp (RGD) recognition sequence. A 40 kD COOH-terminal chymotryptic fragment of fibronectin containing both a heparin-binding region and an alternate (non-RGD) cell-binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin-binding domain, including the CS1 alternate cell-binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits alpha 3, alpha 5, alpha IIb, alpha v, beta 1 and beta 3, but not alpha 4, all bound to trophoblast cells. Antibodies raised against either the beta 1 or beta 3 integrin subunits significantly inhibited fibronectin-mediated outgrowth. These findings demonstrate the key role of the central cell-binding domain of fibronectin in trophoblast adhesion, and suggest four RGD-binding integrins, alpha 3 beta 1, alpha 5 beta 1, alpha IIb beta 3, and alpha v beta 3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation.

In this study, we describe the ultrastructural and immunocytochemical changes that occur in the iris of rats with experimental autoimmune uveoretinitis (EAU). General changes include perivascular infiltration of inflammatory cells, followed by hemorrhage and extensive tissue destruction. Alterations in the iris epithelium were also noted. A breakdown in the blood-iris barrier was demonstrated in some vessels at the peak of inflammation; peroxidase reaction product was seen in the basement membrane and perivascular spaces. We found that, in inflamed iris vessels, endothelial cells and smooth muscle cells become hypertrophic and show increased amounts of synthetic organelles. This finding is similar to our previous observations on endothelial cells and smooth muscle cells in retinal vessels in EAU. In addition, as was reported in the retinal vascular basement membrane in EAU, there is an increase in immunoreactivity of several extracellular matrix molecules in the iris vascular basement membrane; during inflammation, there is a significant increase in immunoreactivity of collagen types I and III, entactin, fibronectin, and laminin. Activated endothelial cells and smooth muscle cells are likely to be involved in the synthesis of certain of these matrix molecules.

This study focused on the effects of ethanol on blastocyst outgrowth and implantation in mice. Blastocysts were exposed to ethanol in Ham's F10 medium and then cultured free of ethanol on fibronectin-coated Petri dishes to assess trophoblast cell adhesion and migration. The time necessary for half of the embryos to outgrow was significantly less (P < 0.05) following treatment with 0.1%, 0.2%, 0.4% or 1.0% (w/v) ethanol for either 5 min or 24 h compared with controls. The rate of trophoblast cell migration was determined by measuring the mean area of outgrowing embryos using an image analysis system. Blastocysts exposed to ethanol for 5 min produced a greater (P < 0.05) average outgrowth area than did stage-matched controls. Acceleration of blastocyst cavitation by ethanol is known to be associated with an increase in the intracellular concentration of calcium. Here, treatment with the calcium ionophore A23187 stimulated (P < 0.05) trophoblast outgrowth and accelerated (P < 0.05) the rate of cell migration. In an attempt to correlate the effect of ethanol on outgrowth in vitro with implantation in utero, cultured blastocysts were either not exposed to ethanol or exposed to 0.1% ethanol for 5 min and transferred 24 h later to the uteri of pseudopregnant dams. The implantation rate (39.4%, n = 376) and the rate of development to term (2.45 pups per mouse, n = 20) were higher (P < 0.05) in mice receiving ethanol-treated embryos compared with those receiving control embryos (20.8%, n = 331; 1.16 pups per mouse, n = 18, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: To determine if cocaine exposure affects human sperm motility, intracellular calcium level, and fertilizing capability., Design and Methods: Human semen samples were treated with 1 to 1,000 microM cocaine hydrochloride for up to 2 hours in vitro. Sperm motion kinematics were measured by computer-assisted semen analysis (CASA). Spermatozoan intracellular calcium was determined by laser cytometry. The sperm fertilizing capability was assessed using the zona-free hamster oocyte penetration test., Results: After a short exposure (15 minutes) to cocaine, the sperm motion kinematic parameters, straight line velocity and linearity, were decreased in the high concentration groups. However, after a longer exposure (2 hours) to cocaine, the differences were no longer significant. Cocaine treatment did not alter spermatozoa intracellular calcium levels. Most importantly, human sperm treated with cocaine at a high concentration were fully capable of penetrating zona-free hamster oocytes., Conclusion: Human spermatozoa acutely exposed to high concentrations of cocaine initially demonstrate a decrease in two motion kinematics, straight line velocity and linearity. However, overall, cocaine exposure had no significant effects on sperm motility and fertilizing capability.

To extend our previous finding that ethanol stimulates murine preimplantation development, we focused in the current study on the cavitation and expansion of the blastocyst. Ethanol stimulation of blastocyst cavitation and expansion was optimal at a concentration of 0.1% and required only a 5-min exposure. Because intracellular levels of calcium were transiently increased in the ethanol-exposed embryos, we determined the effect of directly increasing calcium on the rates of blastocyst cavitation and expansion. Treatment with the calcium ionophore, A23187, altered development much as did ethanol, indicating that ethanol may stimulate preimplantation development by increasing the intracellular calcium concentration. The relationship between intracellular calcium levels and blastocoel volume suggests that morphogenetic events during preimplantation development, particularly cavitation and blastocyst formation, may be regulated through modulation of intracellular calcium levels.

In vitro culture of mouse blastocysts during the period coinciding with implantation has revealed that primary trophoblast cells can adhere and migrate in serum-free medium when provided with certain extracellular matrix components, including fibronectin and laminin. Tightly associated with laminin is the glycoprotein, entactin, that may play an important role in basement membrane assembly and cell attachment. Mouse blastocysts were studied using this in vitro model to determine whether entactin was capable of mediating trophoblast invasive activity. Although entactin has never been shown to promote cell migration, we report here that recombinant entactin supported blastocyst outgrowth in a dose-dependent manner, with a maximal effect at 20-50 micrograms/ml. The ability of trophoblast cells to adhere and migrate on entactin was specifically inhibited by anti-entactin antibody, but not by antibodies raised against laminin. The synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, that contains the Arg-Gly-Asp (RGD) integrin recognition site, reversibly inhibited entactin-mediated blastocyst outgrowth in a dose-dependent manner, but had no effect on laminin-mediated outgrowth. The synthetic peptide, Gly-Phe-Arg-Gly-Asp-Gly-Gln, that comprises the actual RGD-containing sequence within entactin, promoted trophoblast outgrowth when immobilized on the substratum. Furthermore, a mutated recombinant entactin, altered to contain a Glu in place of Asp at the RGD site, provided no trophoblast cell adhesive activity. We conclude that entactin promotes trophoblast outgrowth through a mechanism mediated by the RGD recognition site, and that it may play an important role during invasion of the endometrial basement membrane at implantation.

DNA specific fluorochrome (Hoechst 33342 and 33258) as non-toxic stains, have been widely used to measure cell density and proliferation, detect sperm-egg fusion, and observe the development of pre-implantation embryos. It has been reported that Hoechst 33342 at a concentration of 10 micrograms ml-1 had significant inhibition on embryo cleavage. In this study, we incubated B6D2F1 mouse sperm and eggs with different concentrations of Hoechst 33258, 0, 1.0, 10.0, 20.0, 100 micrograms ml-1. We found that: (1) 100 micrograms ml-1 of H-33258 significantly decreased the sperm motility at 90 min and 4 h. (P less than 0.05), (2) 20 micrograms ml-1 and 100 micrograms ml-1 of Hoechst 33258 significantly inhibited mouse fertilization in vitro (P less than 0.05), and (3) 1.0 micrograms ml-1 and 10.0 micrograms ml-1 Hoechst 33258 had no effect on fertilization rate. But when we pre-incubated sperm at 10 micrograms ml-1 Hoechst 33258 for 90 min, and inseminated oocytes in the medium with same concentration of Hoechst 33258, the embryo cleavage was significantly inhibited.