Richard E. Leach

Extracellular Matrix Composition and Hypoxia Regulate the Expression of HLA-G and Integrins in a Human Trophoblast Cell Line

Abstract During human placentation, extravillous cytotrophoblast cells emerge from chorionic villi contacting the decidua to invade the uterine wall. When isolated from first-trimester placentae, cytotrophoblast cells undergo step-wise differentiation in vitro that recapitulates the phenotypic heterogeneity observed in vivo. We examined a cell line, HTR-8/SVneo, that has been established from human first-trimester cytotrophoblast to determine whether these cells possess some of the unique cytotrophoblast characteristics that have been described previously. Exposure during serum-free culture to hypoxic conditions (2% oxygen concentration) increased HTR-8/SVneo cell proliferation and reduced invasion of a three-dimensional basement membrane (Matrigel). During culture on surfaces coated with individual extracellular matrix proteins, HTR-8/SVneo cells expressed cytokeratin but not the trophoblast-specific major histocompatibility protein, HLA-G. However, HLA-G expression was induced in HTR-8/SVneo cells that contacted Matrigel. Expression of the α5 integrin subunit was relatively unaffected by matrix composition, whereas α1 was up-regulated and α6 was down-regulated after transferring cells to Matrigel. Hypoxia increased α6 and decreased both α1 and HLA-G expression on Matrigel. HTR-8/SVneo cells retain several important characteristics associated with primary cultures of first-trimester human cytotrophoblast cells, including their altered behavior in response to a changing maternal environment.

Reduction of postsurgical adhesion formation in the rabbit uterine horn model with use of hyaluronate/ carboxymethylcellulose gel

OBJECTIVE To assess the efficacy of a bioabsorbable gel for reducing primary postoperative adhesions. DESIGN A randomized, prospective, blinded study. SETTING Academic research environment. ANIMALS Forty-one New Zealand Rabbits. INTERVENTION(S) A chemically modified hyaluronate and carboxymethylcellulose (HA/CMC) gel formulation was applied to a bilateral uterine horn injury. Postoperative adhesions were assessed at a second-look laparoscopy. MAIN OUTCOME MEASURE(S) The uterine horn model was shown to be adhesiogenic, with 29 (70%) of 42 untreated uterine horns found to have adhesions. After gel treatment, 22 (55%) of 40 uterine horns were free of adhesions compared with 12 (30%) of 42 controls. RESULT(S) Animals treated with HA/CMC gel had significantly reduced postsurgical adhesion scores when compared with controls. CONCLUSION(S) Treatment of injured uterine horn with HA/CMC gel resulted in a significant reduction in postoperative surgical adhesions.

Pre-eclampsia and expression of heparin-binding EGF-like growth factor

BACKGROUND Pre-eclampsia is a disorder of pregnancy associated with poor extravillous cytotrophoblast invasion and above-normal rates of apoptosis in the trophoblast. Heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) has strong cytoprotective activity and is an important signalling protein that regulates trophoblast invasion during early placentation. We aimed to establish whether HB-EGF expression is altered in placentae of pre-eclamptic women. METHODS We assessed the expression of HB-EGF mRNA and protein by in-situ hybridisation and immunohistochemical techniques, respectively, in archived placental tissues from pregnancies terminated at around 20 weeks of gestation, and from women delivering between weeks 19 and 35 of gestation with preterm labour, small for gestational age infants, or pre-eclampsia. FINDINGS HB-EGF mRNA and protein were expressed in villous and extravillous cytotrophoblast cells up to week 35 of gestation in placentae from women who delivered preterm. Similar levels of HB-EGF protein were found in the placentae of women who were not in labour. HB-EGF expression was reduced about five-fold (p=0.0001) in pre-eclamptic pregnancies. Fetal growth retardation, which has been linked with shallow trophoblast invasion and moderate apoptosis, was associated with placentae expressing intermediate levels of HB-EGF. INTERPRETATION In pre-eclampsia, deficient HB-EGF signalling during placental development could impair trophoblast survival, differentiation, and invasion, leading to poor placental perfusion and hypertension.

Reduction of postoperative adhesions in the rat uterine hornmodel with poloxamer 407

Adjuvants shown to have significant adhesion prevention capabilities have been based on barrier separation of deperitonealized surfaces. We investigated the adhesion prevention properties of poloxamer 407. This polyol, in the proper concentration, is fluid at room temperature and becomes a firm gel at body temperature. The prevention of postoperative adhesions was evaluated in the rat uterine horn model. Twenty-two rats underwent bilateral surgical injury to the uterine horn and the parietal peritoneum. A random side was treated with a 30% solution of poloxamer 407. Twenty-one days later the animals were sacrificed and evaluated for the presence of adhesions. A highly significant reduction in adhesion formation was documented on the treated side.

Unexplained infertility: Evaluation of the luteal phase; results of the National Center for Infertility Research at Michigan

OBJECTIVE To evaluate the luteal phase in women with rigorously defined unexplained infertility. DESIGN Prospective study. SETTING National Center for Infertility Research at Michigan. PATIENT(S) Evaluation of 1,885 women with infertility identified 12 women who met the rigorously defined criteria for unexplained infertility: [1] infertility of > or = 24 months duration, with no male factor, anatomic-functional disorders of the reproductive tract, or immunologic infertility; [2] normal body mass index (BMI); [3] ovulatory cycles ranging from 26 to 32 days; [4] normal luteal phase determined by endometrial biopsy; and [5] normal baseline hormonal profile. Controls (n = 12) were healthy, parous women with normal ovulatory cycles, normal hormonal screen, and were matched for age and BMI to patients. MAIN OUTCOME MEASURE(S) Pattern of follicular growth rate and luteal phase hormonal profile. RESULT(S) Women with unexplained infertility did not differ in menstrual cycle characteristics, follicular growth rate or mean preovulatory follicle diameter, or endometrial biopsy dating. The mean levels of P tended to be lower in the unexplained infertility group throughout the luteal phase, but only the midluteal interval reached statistical significance. Luteal phase mean integrated P or urinary PDG levels of unexplained infertility women did not differ from those of fertile controls. The ratio of integrated E2:P also was significantly greater in women with unexplained infertility than in fertile controls. CONCLUSION(S) Women with rigorously defined unexplained infertility have subtle hormonal anomalies during the luteal phase when compared with fertile controls.

A comparative evaluation of Poloxamer 407* and oxidized regenerated cellulose (Interceed [TC7]*) to reduce postoperative adhesion formation in the rat uterine horn model†‡

OBJECTIVES To determine if Poloxamer 407 (poloxamer, Pluronic F-127; BASF Wyandotte Corp., Parsippany, NJ) is as effective as Interceed(TC7) (Ethicon, a Johnson and Johnson company, Sommerville, NJ) in preventing postoperative adhesion formation using the rat uterine horn model and to determine if the presence of blood or lactated Ringers solution affects the effectiveness of Poloxamer 407. DESIGN, SETTING, PARTICIPANTS Sprague-Dawley white rats, weighing 225 to 250 g in a conventional laboratory setting. The left or right sidewall was randomly assigned to receive no treatment (control), Interceed(TC7), or poloxamer. INTERVENTIONS Each uterine horn and ipsilateral sidewall was subjected to a standardized lesion of denudation. To evaluate the barrier agents in the presence of blood, a sidewall vessel was ligated and a thrombus allowed to form. To evaluate the effectiveness of lactated Ringers solution, 10 mL was injected intraperitoneally after abdominal closure. MAIN OUTCOME MEASURES Degree of adhesion formation was evaluated 14 days after surgery. RESULTS The adhesion score for the poloxamer-treated animals was significantly lower than its control. Interceed(TC7) did not reduce adhesion formation as compared with its control. In animals that received both poloxamer and Interceed(TC7) on either side, the poloxamer-treated sides had a significantly lower adhesion score than Interceed(TC7)-treated sides. The presence of blood and lactated Ringers reduced the adhesion-reducing properties of poloxamer. CONCLUSION In this model, poloxamer is more effective than Interceed(TC7) in the prevention of postoperative adhesion formation. These findings also suggest that the presence of blood compromises the effectiveness of poloxamer to prevent postoperative adhesion formation, therefore requiring complete hemostasis before poloxamers application. Lactated Ringers instillation was ineffective in reducing adhesion formation on the control or poloxamer-treated sidewall.

Fibrinolytic parameters in women undergoing ovulation induction

OBJECTIVE The purpose of this study was to evaluate the effect of elevated levels of circulating estradiol on the clotting and fibrinolytic system in patients undergoing controlled ovarian hyperstimulation. STUDY DESIGN Fifty-two patients undergoing controlled ovarian hyperstimulation with human menopausal gonadotropins or urofollotropin were asked to participate. Blood for hemostasis parameters was obtained on the days that patients returned for estradiol sampling. Sample days were identified as cycle days 1 to 5 (baseline), 6 to 9, and 10 to 14. Each factor was analyzed by repeated-measures analysis of variance and correlation analysis. RESULTS A significant decline was observed for tissue plasminogen activator antigen and plasminogen activator inhibitor type 1 activity from baseline to cycle days 10 to 14. As serum estradiol levels increased throughout each phase (maximum mean estradiol 739.8 pg/ml), a significant linear decrease was observed for both tissue plasminogen activator antigen and plasminogen activator inhibitor type 1 activity, whereas thrombin-antithrombin III complexes did not change significantly. A significant positive correlation was also observed for plasminogen activator inhibitor activity and tissue plasminogen activator antigen level over all cycle days examined. CONCLUSION Down-regulation of the fibrinolytic system was observed as estradiol levels increased. However, thrombin formation did not change, thus suggesting that elevated circulating estradiol alone does not predispose to a thromboembolic event.

Selective embryo reduction in a heterotopic pregnancy using potassium chloride injection resulting in a hematosalpinx

OBJECTIVE To incorporate conservative management of a heterotopic pregnancy using injection of KCl into the ectopic pregnancy (EP). DESIGN A retrospective case report. SETTING A patient referred to an academic institution in the division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, incorporating the ultrasound (US) and operating room facilities. INTERVENTION(S) Using US as a guide, KCl was injected into the chorionic cavity of an EP. MAIN OUTCOME MEASURE(S) Resolution of ectopic gestational tissue with resultant hematosalpinx requiring exploratory laparotomy. Uncomplicated prenatal course of intrauterine pregnancy. RESULT(S) Injection of KCl into the chorionic cavity of EP resulting in resolution of gestational tissue but complicated by hematosalpinx in the fallopian tube. CONCLUSION(S) Selective embryo reduction of a tubal heterotopic pregnancy remains a viable therapeutic option.

Selective embryo reduction of an interstitial heterotopic gestation.

A case of combined interstitial and intrauterine pregnancy is described. The sonographic finding of a thick myometrial bridge separating a twin pregnancy may be unique to heterotopic cornual pregnancies and is illustrated. The method of selective embryo reduction with potassium chloride to terminate the interstitial pregnancy without damage to the intrauterine pregnancy is also described. The intrauterine pregnancy continued successfully to term.

Elevated early follicular gonadotropin levels in women with unexplained infertility do not provide evidence for disordered gonadotropin-releasing hormone secretion as assessed by luteinizing hormone pulse characteristics

OBJECTIVE To determine whether women with rigorously defined unexplained infertility demonstrated altered GnRH secretion, as reflected by serum LH secretion patterns. DESIGN Prospective observational study. SETTING National Center for Infertility Research at Michigan. PATIENT(S) Nine women with rigorously defined unexplained infertility and 11 healthy, parous age-matched control women.Gonadotropin-releasing hormone (25 ng/kg) as a bolus injection. MAIN OUTCOME MEASURE(S) Daytime pulse patterns of LH secretion measured every 10 minutes; mean serum concentrations of LH, FSH, E(2), P, PRL, and cortisol; and response to a physiologic dose of GnRH in the early follicular, late follicular, mid-luteal, and late luteal phases of the same menstrual cycle. RESULT(S) Serum LH pulse frequency and pulse amplitude and LH secretion in response to a physiologic bolus of GnRH were not significantly different in unexplained infertility patients at any phase of the cycle. Luteinizing hormone pulse frequency and amplitude, as well as response to GnRH, varied significantly across the cycle. Mean early follicular serum LH and FSH concentrations were significantly higher in unexplained infertility patients than in fertile control subjects (LH: 5.31 +/-.51 vs. 4.03 +/-.33 [mIU/mL +/- SEM]; FSH: 5.81 +/-.63 vs. 3.80 +/-.45) but were not different at any other phase of the cycle. CONCLUSION(S) These data do not support the hypothesis that unexplained infertility is caused by an abnormality in pulsatile GnRH secretion or abnormal pituitary sensitivity to GnRH. However, the results are consistent with a difference in negative feedback from the ovary to the pituitary in unexplained infertility patients that is suggestive of diminished ovarian reserve.