Quantifying the effect of weathering on acute oil toxicity using the PETROTOX model.

Following accidental release into marine environments, crude oil progressively weathers, influencing composition, fate, and toxicity. However, published studies draw conflicting conclusions on the effects of oil weathering on ecotoxicity. Using the PETROTOX model, this study characterized the effect of weathering on acute oil toxicity for four aquatic species. Results indicated that predicted acute toxicity decreased with increased oil weathering, due to reductions in overall concentrations and bioavailability of hydrocarbon constituents.

In vitro developmental potential of macaque oocytes, derived from unstimulated ovaries, following maturation in the presence of glutathione ethyl ester.

BACKGROUND: The inadequacies of oocyte in vitro maturation (IVM) systems for both non-human primates and humans are evidenced by reduced fertilization and poor embryonic development, and may be partly explained by significantly lower glutathione (GSH) contents compared with in vivo matured (IVO) oocytes. As this influence has not been fully explored, this study investigated the effect of the GSH donor, glutathione ethyl ester (GSH-OEt), on the IVM and development of macaque oocytes as a model of human oocyte IVM. METHODS: Macaque oocytes derived from unstimulated ovaries were cultured in mCMRL-1066 alone or supplemented with 3 or 5 mM GSH-OEt. In vitro matured oocytes were subjected to the GSH assay, fixed for the assessment of spindle morphology or prepared ICSI. Embryo development of zygotes cultured in mHECM-9 was assessed up to Day 9 post-ICSI. RESULTS Supplementation of the maturation medium with GSH-OEt significantly increased oocyte maturation and normal fertilization rates compared with control oocytes, but only 5 mM GSH-OEt significantly increased the oocyte and cumulus cell GSH content. Confocal microscopy revealed significant differences in the spindle morphology between IVO and control in vitro matured metaphase II oocytes. Oocytes matured with 5 mM GSH-OEt exhibited spindle area and spindle pole width similar to that seen in the IVO oocyte. While no significant differences were observed in blastocyst rates, addition of 3 mM GSH-OEt during IVM significantly increased the proportion of embryos developing to the 5-8 cell stage while 5 mM GSH-OEt significantly increased the proportion of morula-stage embryos compared with controls. CONCLUSIONS: Supplementation of the IVM medium with GSH-OEt promotes better maturation and normal fertilization of macaque oocytes compared with non-supplemented medium. However, further improvement of the primate oocyte IVM culture system is required to support better blastocyst development of oocytes derived from unstimulated ovaries.

Oocyte glutathione and fertilisation outcome of Macaca nemestrina and Macaca fascicularis in in vivo- and in vitro-matured oocytes.

Fertilisation and development of IVM non-human primate oocytes is limited compared with that of in vivo-matured (IVO) oocytes. The present study describes the IVM of macaque oocytes with reference to oocyte glutathione (GSH). Timing of maturation, comparison of IVM media and cysteamine (CYS) supplementation as a modulator of GSH were investigated. A significantly greater proportion of oocytes reached MII after 30 h compared with 24 h of IVM. Following insemination, IVM oocytes had a significantly lower incidence of normal fertilisation (i.e. 2PN = two pronuclei and at least one polar body) and a higher rate of abnormal fertilisation (1PN = one pronucleus and at least one polar body) compared with IVO oocytes. Immunofluorescence of 1PN zygotes identified incomplete sperm head decondensation and failure of male pronucleus formation as the principal cause of abnormal fertilisation in IVM oocytes. The IVO oocytes had significantly higher GSH content than IVM oocytes. Cumulus-denuded oocytes had significantly lower GSH following IVM compared with immature oocytes at collection. Cysteamine supplementation of the IVM medium significantly increased the GSH level of cumulus-intact oocytes and reduced the incidence of 1PN formation, but did not improve GSH levels of the denuded oocyte. Suboptimal GSH levels in macaque IVM oocytes may be related to reduced fertilisation outcomes.

Developmental potential of bovine oocytes following IVM in the presence of glutathione ethyl ester.

Glutathione (GSH) is synthesised during oocyte maturation and represents the oocyte's main non-enzymatic defence against oxidative stress. Inadequate defence against oxidative stress may be related to poor embryo quality and viability. In the present study, bovine oocytes were matured in vitro in the presence of GSH ethyl ester (GSH-OEt), a cell permeable GSH donor, and its effects on subsequent fertilisation and embryo development were assessed. GSH-OEt significantly increased the GSH content of IVM oocytes without affecting fertilisation or Day 3 cleavage rates. Maturation in the presence of GSH-OEt did not significantly increase the blastocyst rate compared with control oocytes. However, 5 mM GSH-OEt treatment resulted in significantly higher blastocyst total cell number. The GSH level of IVM oocytes was significantly decreased in the absence of cumulus cells and when cumulus-oocyte complexes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine oocytes and restored the rate of normal fertilisation, but not embryo development, to levels seen in control oocytes. Thus, GSH-OEt represents a novel approach for effective in vitro elevation of bovine oocyte GSH and improvement in blastocyst cell number.

Pregnancy-associated plasma protein A in human ovarian follicular fluid.

Human ovarian follicular fluid contains pregnancy-associated plasma protein A (PAPP-A) immunoreactivity as detected by RIA. This PAPP-A was found to be stable to repeated freeze-thaw cycles and not removed by dialysis. Further characterization showed that ovarian follicular PAPP-A bound reversibly to heparin-Sepharose. On gel chromatography follicular PAPP-A coeluted with radioiodinated PAPP-A and pregnancy serum PAPP-A which was determined to have an apparent mol wt of 820,000. In the RIA, serial dilutions of high molecular weight heparin-Sepharose binding proteins gave parallel displacement curves to pooled late pregnancy serum and to the International Reference Preparation, WHO 78/610. All the human ovarian follicular fluids tested had PAPP-A concentrations between 0.317-1.595 IU/liter. The relationship between follicular content of PAPP-A and follicular volume was best expressed by the exponential relationship, y = 164.3 e0.117x. Therefore, ovarian follicular fluid PAPP-A has many physico-chemical similarities to and shares immunological identity with pregnancy-derived PAPP-A. As this pregnancy-associated glycoprotein cannot be detected in the circulation of normal non-pregnant adults, its presence within the Graafian follicle is believed to be of intrafollicular origin for the maintenance of proteolytic homeostasis.

In-vitro secretion of prostanoids by placental villous cytotrophoblasts in pre-eclampsia.

Villous trophoblasts isolated from term placentae of normal pregnancies, and pregnancies complicated by chronic hypertension or pre-eclampsia, were examined over 7 days in primary culture. Low levels of prostaglandin E2 and prostacyclin (measured as 6-keto prostaglandin Fl alpha) were secreted by trophoblast cells from all three clinical groups. Secretion was maximal at day 1 and decreased exponentially thereafter. Thromboxane secretion also fell sequentially from day 1. Thromboxane secretion by pre-eclamptic trophoblasts was three to four times that of cells from normal or chronically hypertensive subjects. Prostanoid secretion by isolated cultured cytotrophoblasts was not dependent on aggregation or morphological alteration, nor related to changes in progesterone or human chorionic gonadotrophin production. Because the local maternal circulation is exposed to substances secreted by this cell population, thromboxane could be the trigger for vasoconstriction and coagulation found within the maternal uteroplacental circulation in pre-eclampsia.

Potential role of pregnancy-associated plasma protein-A in human reproduction.

By radioimmunoassay, pregnancy-associated plasma protein-A (PAPP-A) was undetectable in matched follicular and luteal phase serum samples (n = 17) or in the peripheral circulation of normal males (n = 17). However, seminal plasma (91.5%), cervical mucus (100%) and pre-ovulatory follicular fluid (99.6%) were consistently PaPP-A positive. In addition to PAPP-A, four circulating protease inhibitors (PIs) were detected in pooled seminal plasma whereas pooled follicular fluid contained an additional six. Follicular concentrations of serum PIs were inversely related to molecular size. By contrast, PAPP-A formed a positive concentration gradient across the blood-reproductive tract barrier suggesting PAPP-A production within the reproductive tract. A minor proportion (1.7%) of ejaculated spermatozoa were coated with PAPP-A, as demonstrated by direct immunofluorescence. Since PAPP-A specifically inhibits leucocyte elastase, it is suggested that PAPP-A coated spermatozoa were "selected" to overcome localized phagocytic-proteolytic degradation. The physiological significance of these findings are discussed in relation to human reproduction.

Monitoring of postimplantation embryo viability by measurement of PAPP-A, SP1, and hCG.

After successful in vitro fertilization and embryo transfer, chorionic gonadotropin, pregnancy-specific beta 1-glycoprotein, and pregnancy-associated plasma protein-A were measured in serum samples collected serially from 21 patients. While 14 pregnancies, including one twin pregnancy, progressed successfully to term, the remaining seven pregnancies failed during the first half of gestation. This latter group consisted of three tubally implanted, one anembryonic, and three spontaneously aborted pregnancies. Circulating levels of hCG, SP1, and PAPP-A in the patient with an anembryonic pregnancy were within normal limits. Similarly, 90.5% of the serum samples obtained from women with tubal pregnancies showed hCG levels within normal limits. By contrast, only two of these samples had detectable PAPP-A, of which only one was within normal limits. Of the samples obtained from the patients who spontaneously aborted, including one patient with normal ultrasonic findings up to 48 hours prior to the event, 85.7% had PAPP-A concentration below the 10th percentile, whereas only 16.7% of these samples showed depressed hCG levels. These data suggest that PAPP-A measurement has great clinical potential in the management of compromised early pregnancies.

Pregnancy-associated plasma protein-A and placental protein 5 in human ovarian follicular fluid.

By sensitive and specific radioimmunoassays PAPP-A and PP5 were detected in follicular aspirates obtained from women undergoing ovarian hyperstimulation for oocyte harvest prior to in vitro fertilization and embryo transfer. Follicular and pregnancy-derived PAPP-A were immunologically and physicochemically indistinguishable. Similarly, pregnancy- and nonpregnancy-derived PP5 were immunologically indistinguishable. However, in addition to the 18- and 36-K species, a larger species having a molecular size greater than 140K was found in the follicular fluid. Mean follicular PAPP-A and PP5 concentrations were 727 mIU/L and 1376 mAU/L, respectively, with no significant correlation between follicular PAPP-A, PP5, and steroid concentrations. There was, however, a significant but negative relationship with follicular volume. Preliminary in vitro studies indicated that both proteins were synthesized by granulosa cells in preparation for follicular rupture. Follicular PP5, like antithrombin III, interacted reversibly with heparin and thrombin affinity matrices, suggesting a potential biological role as a follicular anticoagulant, whereas PAPP-A, a specific and potent inhibitor of leukocyte elastase, contributes to the maintenance of proteolytic homeostasis and the protection of spermatozoa and embryo against proteolytic attack originating from the maternal leukocytes.

Pregnancy-associated plasma protein-A and placental protein 5 in human seminal plasma.

Human seminal plasma contains two glycoproteins which are physiochemically and immunologically indistinguishable from pregnancy-associated plasma protein-A and placental protein 5. Seminal concentrations of both glycoproteins did not correlate with clinical assessment of semen quality. Furthermore, analysis of split ejaculates indicated a nontesticular origin for both proteins, which are possibly secreted into the distal portions of the tract by the accessory glands (prostate gland and seminal vesicles). The physiological significance of these findings has yet to be determined. However, it is suggested that pregnancy-associated plasma protein-A, a known potent inhibitor of leukocyte elastase, protects the deposited sperm against proteolytic attack originating from the localized leukocyte reaction within the female reproductive tract, thus contributing towards sperm survival within this immunologically hostile environment and enabling fertilization to occur.

Feminization in gonadal dysgenesis associated with ovarian gonadoblastoma.

A case of gonadal dysgenesis in a phenotypic female with XY genotype associated with ovarian gonadoblastoma is reported. Endocrinologic studies suggest that the normal female secondary sex characteristics could be due to estrogen production from luteal intersitial cells.

Twin pregnancy suspected before implantation.

A case is reported in which the diagnosis of twin pregnancy was suspected by the appearance of 2 follicles on ultrasound examination and by high levels of plasma estradiol and progesterone.

A safe method of amniocentesis for lecithin/sphingomyelin determination in late pregnancy using ultrasound.

Two hundred consecutive amniocenteses for lecithin/sphingomyelin (L/S) determination in late pregnancy are reviewed. These procedures were performed under ultrasonic control before induction of labor or elective cesarean section in a prospective study to determine a safe method for aminocentesis. Complications included 3 failures, 14 blood-stained samples, 2 cases of labor occuring within 24 hours after amniocentesis, and 1 maternal abdominal wall hematoma. There was no cases of perinatal morbidity or mortality. It is proposed that this is a safe method of abdominal amniocentesis for L/S determination in late pregnancy.

Assessment of ovulation by ultrasound and plasma estradiol determinations.

The timing of ovulation was studied daily during the periovular period in 19 women attending an artificial insemination clinic during 24 menstrual cycles; B-mode ultrasound examination and plasma estradiol (E2) determinations were used. The maximum diameters of the ovarian follicles and the peak plasma E2 determinations all occurred within 2 days prior to and including the day of ovulation, as determined by conventional means, in the 12 normal cycles studied. The average values on the day prior to ovulation (day -1) for maximum follicle size and peak E2 determination were 2.5 cm and 1660 pmole/liter, respectively. Two definite ultrasonic patterns were noted after ovulation: The follicle either disappeared or filled with internal echoes. It is concluded from the preliminary study that ultrasound examination and plasma E2 determinations are equally effective in predicting the time of ovulation.

Effects of clomiphene administration on ovarian function as measured by estradiol and ultrasound.

The growth and rupture of the graafian follicle were studied in 23 women during 40 cycles (20 spontaneous, 20 clomiphene-induced) by the estimation of plasma estradiol levels and ultrasound scan. The mean preovulatory estradiol peak level was 415 pg/ml in spontaneous cycles and 626 pg/ml after clomiphene administration when one follicle was present. Ultrasonic examination revealed the presence of more than one developing follicle in one of 20 spontaneous cycles, and in 11 of 20 cycles after clomiphene treatment. The determination of follicle size and number by ultrasound scan during ovulation induction by the routinely used starting dosage of clomiphene has revealed a previously unrecognized incidence of ovarian overstimulation, and may allow the rationalization of ovulation induction regimens.

Treatment of luteal phase inadequacy with bromocriptine.

A group of 15 women diagnosed as having luteal phase inadequacy (LPI) and a history of 1--2 years of infertility were treated with bromocriptine continuously for 4 months. Plasma levels of prolactin, progesterone, 17 alpha-hydroxy-progesterone and estradiol were measured by radioimmunoassay in a control month and each of the treatment months. There was no significant improvement in the levels of the steroids over the 4-month period, although the prolactin levels were depressed, and 2 subjects became pregnant during the study.

Clinical outcomes of pregnancies achieved by microinsemination by sperm transfer.

Micromanipulative techniques such as microinsemination by sperm transfer are evolving as treatment for couples suffering from some forms of male factor infertility where previously donor insemination was the only option. Being a relatively new technology, the numbers of microinsemination by sperm transfer pregnancies that have progressed to term are still small. However, at this time, it would seem that outcome in terms of antenatal complications, gestation length, and birth weight is no different from that seen with conventional IVF. Continuing review of the rate of congenital malformation in microinsemination by sperm transfer progeny seems warranted.

Elevated luteal phase estradiol:progesterone ratio in mice causes implantation failure by creating a uterine environment that suppresses embryonic metabolism.

Failure of implantation after in vitro fertilization is sometimes associated with elevated estradiol (E2):progesterone (P) ratios in the early luteal phase. Using the mouse to study the causes of the contragestational actions of elevated E2:P ratios, it was confirmed that administration of E2 (40 to 80 ng) before implantation inhibited implantation and 0.4 mg of P totally reversed the effect of 40 ng E2. Fluids collected from the uterine lumen of E2-treated (40 ng) mice significantly inhibited the incorporation of 3H-uridine by mouse blastocysts in vitro, whereas fluids from mice treated with E2 (40 ng) and P (0.4 mg) together had no inhibitory effect. In contrast to these contraceptive effects of high E2, lower levels of E2 (5 and 20 ng) caused an increase and no change to the implantation rate, respectively, and fluids from mice treated with E2 (20 ng) significantly enhanced blastocyst metabolism. Concomitant administration of P to these low dose E2 mice enhanced these effects. Thus, a large elevation of the E2:P ratio inhibited implantation and created a uterine lumenal milieu that suppressed embryonic metabolism. These effects could be reversed and implantation re-established by the correction of the E2:P ratio by exogenous P.

Monitoring of postimplantation embryo viability following successful in vitro fertilization and embryo transfer by measurement of placental proteins.

Serum levels of pregnancy-associated plasma protein A (PAPP-A), human chorionic gonadotropin (hCG), and pregnancy-specific beta 1-glycoprotein (SP1) were measured in 21 women after successful in vitro fertilization and embryo transfer. Of the 21 pregnancies, 14, including 1 twin gestation, progressed successfully to term. The remaining seven, composed of tubal (n = 3), anembryonic (n = 1), and spontaneously aborted (n = 3) pregnancies, failed during the first half of pregnancy. Placental protein measurement was of no diagnostic value in the detection of anembryonic pregnancy. Similarly, measurement of hCG and SP1 could not readily distinguish tubal ectopic from normal intrauterine pregnancies. By contrast, the predictive value (38.9%) of a depressed PAPP-A level in conjunction with superior diagnostic sensitivity (70%) and relative risk factor (23.6) proved to be of greater diagnostic value in this potentially lethal condition. In the absence of ultrasonography, the biochemical diagnostic indices were comparable in the prediction of spontaneous abortion. However, in the presence of a live fetus, PAPP-A levels were consistently depressed (sensitivity, 91.7%) many weeks before pregnancy demise. The relative risk factor of depressed PAPP-A levels was 29 times greater than the risk associated with a depressed hCG level. These findings further demonstrate the potential diagnostic value of PAPP-A measurement for monitoring postimplantation embryo viability.

Assessment of ovulation by ultrasound and estradiol levels during spontaneous and induced cycles.

Both Graafian follicle growth and subsequent ovulation were studied in 45 menstrual cycles of 28 patients by the estimation of plasma estradiol levels and by the measurement of follicle size and number by ultrasound. Twenty spontaneous ovulatory cycles were studied as controls compared with twenty cycles in which ovulation was induced by clomiphene and five cycles in which ovulation was induced by human pituitary gonadotropin. The means of the peak estradiol levels during the cycles in which one follicle was present were 1553.1 +/- 87.8 pmoles/liter in the spontaneous cycles and 2296.8 +/- 163.4 pmoles/liter in the clomiphene-treated cycles. Ultrasound was shown to be complementary to endocrine profiles because the number and diameter of Graafian follicles could be measured accurately by this technique.