Does early morphology provide additional selection power to blastocyst selection for transfer?

Routine early developmental parameters are widely used in IVF centres to evaluate embryo development and fresh single-blastocyst transfer currently seems superior to single-embryo transfer. Would early morphological parameters help to choose the single blastocyst to be transferred, thereby improving the chances of implantation and live birth rate? This prospective observational study analysed the individual outcomes of 2617 embryos from 511 IVF couples scheduled for a single-blastocyst transfer. Embryo and blastocyst scores were constructed. There was a clear relationship between the kinetics and morphology of blastocysts and further implantation and live birth rate. There was a limited predictive value of embryo score with regard to blastocyst development and growth kinetics. Implanted and non-implanted blastocysts showed similar embryo scores. Thus usual morphological parameters on days 1 and 2 seem to have no additional value in indicating the right blastocyst to transfer. Non-invasive approaches might be helpful to increase the chances of implantation in the future.

Single Day 2 embryo versus blastocyst-stage transfer: a prospective study integrating fresh and frozen embryo transfers.

BACKGROUND: Whether extended culture allowing selection of embryos with high development potential has any advantage over cleavage-stage embryo transfer remains a matter of debate. Among the currently unsolved questions, the cumulative delivery rate resulting from fresh and frozen embryo transfers needs to be taken into account in both strategies. The aim of our study was, therefore, to compare the efficacy of single embryo transfer either on Day 2 or on Day 5/6 combining fresh and frozen embryo transfers. METHODS: A prospective study including 478 couples assigned on a voluntary basis to undergo elective single embryo transfer (eSET, n = 243) on Day 2 or single blastocyst transfer (SBT, n = 235) on Day 5/6 was performed. The primary outcome measurement was the cumulative delivery rate including fresh and frozen-thawed cycles in both groups. RESULTS: The delivery rate per cycle following fresh embryo transfer was significantly higher in the SBT group compared with the eSET group (P < 0.01). Conversely, frozen embryo and/or blastocyst transfers tended to result in a higher number of deliveries in the eSET compared with the SBT group. Altogether, the cumulative delivery rate per couple, including fresh and frozen embryo transfers, was similar between the two groups (37.9% versus 34.2% in the SBT and eSET groups, respectively). CONCLUSIONS: The observed cumulative delivery rates in this study do not allow us to take a position in favor of SBT or eSET. An improvement in blastocyst cryopreservation may change this attitude.

Gene expression in human cumulus cells: one approach to oocyte competence.

BACKGROUND: Dialogue between the oocyte and cumulus cells is essential for oocyte maturation. A prospective laboratory research project was designed to evaluate transcription of specific genes in cumulus cells harvested before intracytoplasmic sperm injection from pre-ovulatory follicles, according to individual oocyte nuclear maturity and developmental competence. Genes were chosen because their expression was induced by the LH peak [Steroidogenic Acute Regulatory protein (STAR), Cyclooxygenase 2 (COX2 or PTGS2), Amphiregulin (AREG)] or because they were involved in oocyte lipidic metabolism [Stearoyl-Coenzyme A Desaturase 1 and 5 (SCD1 and SCD5)] or in gap-junctions [Connexin 43 (CX43 or GJA1)]. METHODS: mRNA levels in cumulus cells were assessed by real-time PCR. RESULTS: Expression levels of all genes investigated, except Cx43, were increased after resumption of meiosis. Nuclear maturation was thus associated with increased expression of STAR, COX2, AREG, SCD1 and SCD5 by cumulus cells. When considering only cumulus associated with metaphase II oocytes, gene expression was independent of morphological status at Day 2. In contrast, transcript levels were lower and distributed over a narrower range in cumulus enclosing oocytes achieving blastocyst development at Day 5/6 than in cumulus enclosing oocytes unable to develop beyond the embryo stage. CONCLUSION: Further developmental potential from embryo to blastocyst stage was associated with lower expression in a narrow range for these genes.

Limited value of morphological assessment at days 1 and 2 to predict blastocyst development potential: a prospective study based on 4042 embryos.

BACKGROUND: Non-invasive and routine developmental markers are available to select the most viable embryo; however their respective values in terms of blastocyst development potential remain difficult to distinguish. METHODS: During this prospective study, the sequential growth of 4042 embryos individually cultured from day 1 to day 5/6 was recorded. Pronuclear morphology on day 1, and early cleavage, cell number and fragmentation rate on day 2 were evaluated for each zygote. Additionally, blastocyst transfers were analysed with regard to their implantation ability and early embryo development parameters. RESULTS: Once adjusted to each other, each of the four parameters remained related to blastocyst development. Early cleavage and cell number on day 2 were the most powerful parameters to predict the development of a good morphology blastocyst at day 5. Moreover, whereas transfers of a good morphology blastocyst were associated with high implantation and live birth rates, parameters of early development were not helpful in predicting their implantation ability. CONCLUSIONS: The combination of all four parameters allowed the prediction of blastocyst development with an area under the receiver operating characteristics curve of 0.688, which represents a fairly low prediction of embryo viability. Such results indicate that it is necessary to search for additional criteria, including the ability of the blastocyst to develop.

[Sequential assessment of individually cultured embryos in IVF]. / Suivi individuel des embryons en FIV.

OBJECTIVE: It is a challenge for IVF centers to propose a method to select the most viable embryo to transfer, thereby minimizing the risk of multiple births. In this study, a prospective investigation was made to determine if non-invasive developmental markers on day 1 combined to conventional evaluation on day 2 can predict in vitro blastocyst development. PATIENTS AND METHODS: A total of 4190 individually cultured embryos from patients undergoing IVF/ICSI treatment at the Tours University Hospital Center from January 2002 to December 2004 were included. Individual embryos were cultured in sequential media in microdrops under mineral oil from j1 to j5/j6 allowing to record their sequential growth until the blastocyst stage. RESULTS: The results showed a significant positive relationship between pattern 0 zygote, early cleavage, 4 cells embryos with < 20% fragmentation on day 2 and the rate of blastocyst development on day 5 (P < 0.05). In our hands, zygote pattern does not bring additional benefit to better select embryo. DISCUSSION AND CONCLUSION: Zygote and early cleavage assessments on day 1, morphological appearance on day 2 are some other parameters related individually to blastocyst development on days 5 and 6. These parameters can be used collectively to establish a predictive in vitro sequential embryo assessment model for routine use in IVF clinics.

Efficacy of blastocyst transfer after implantation failure.

Clinicians who treat unsuccessful couples despite repeated transfers of good quality embryos face a challenge. Among the various strategies that have been described, embryo transfer at the blastocyst stage has been postulated to improve implantation. A prospective non-randomized analysis was performed in 276 IVF patients who failed to conceive after at least two early embryo transfers of at least two grade 1-2 embryos per cycle. For the next attempt, couples chose between day 2 embryo transfer (D2 group; n = 147) and day 5/6 blastocyst transfer (D5/D6 group; n = 129) before starting the following attempt. Embryo quality was assessed and results were expressed as clinical pregnancy, live birth and implantation rates per cycle. Embryo grade 1 number was similar between both groups, whereas mean embryo score of the whole cohort was slightly higher in the D2 group. The live birth rates per cycle (27.9 versus 19.7%) and implantation rates per cycle (25.4 versus 12.4%) were higher in the D5/D6 group compared with the D2 group. Improved embryo selection and uterine receptivity may explain the additional benefit of embryo transfer at the blastocyst stage for couples with repeated implantation failures.

Multiple interactions of cimetidine and probenecid with valaciclovir and its metabolite acyclovir.

The effects of probenecid and cimetidine on the pharmacokinetics of valaciclovir and its metabolite acyclovir have been investigated. Twelve healthy male volunteers participated in this open single-dose study with a four-way-crossover randomized and balanced design. At the first of four administrations, volunteers in four groups received 1 g of valaciclovir alone, valaciclovir with 1 g of probenecid, valaciclovir with 800 mg of cimetidine, or valaciclovir with a combination of probenecid and cimetidine. At three subsequent administrations, drug regimens were alternated among groups so that each group received each regimen. Probenecid and cimetidine increased the mean maximum concentrations in serum (C(max)) of valaciclovir by 23 and 53% and the areas under the concentration-time curves (AUC) for valaciclovir by 22 and 73%, respectively; probenecid and cimetidine also increased the mean acyclovir C(max) by 22 and 8% and its AUC by 48 and 27%, respectively. The combination had a greater effect than either drug alone. Their effects may be due to competitive inhibition of membrane transport of valaciclovir and acyclovir in the liver and kidney. Neither cimetidine nor probenecid affected the absorption of valaciclovir. Both probe drugs reduced the rate of valaciclovir metabolism but not its extent. These pharmacokinetic modifications did not affect the tolerability of valaciclovir.

Influence of cirrhosis on lamotrigine pharmacokinetics.

AIMS: Lamotrigine, an antiepileptic drug, is cleared from the systemic circulation mainly by glucuronidation. The possibility of changes in the pharmacokinetics of lamotrigine in plasma owing to hepatic dysfunction has been evaluated. METHODS: Thirty-six subjects, including 24 patients with various degrees of liver cirrhosis and 12 healthy volunteers received a single 100 mg dose of lamotrgine. Blood samples were taken for 7 days in all subjects, except nine with severe cirrhosis, who had a 29 day blood sampling period. RESULTS: The pharmacokinetics of lamotrigine were comparable between the patients with moderate cirrhosis (corresponding to Child-Pugh grade A) and the healthy subjects. Plasma oral clearance mean ratios (90% confidence interval) in patients with severe cirrhosis without or with ascites (corresponding, respectively, to Child-Pugh grade B and C) to healthy subjects were, respectively, 60% (44%, 83%) and 36% (25%, 52%). Plasma half-life mean ratios (90% confidence interval) in these two patient groups to healthy subjects were, respectively, 204% (149%, 278%) and 287% (202%, 408%). CONCLUSIONS: Lamotrigine administered as a single oral dose of 100 mg was well tolerated in all groups. Initial, escalation and maintenance doses should generally be reduced by approximately 50 or 75% in patients with Child-Pugh Grade B or C cirrhosis. Escalation and maintenance doses should be adjusted according to clinical response.

Pharmacokinetics of oral acyclovir in neonates and in infants: a population analysis.

Acyclovir is approved for the treatment of herpes simplex virus (HSV) and varicella-zoster virus (VZV) infections in children by the intravenous and oral routes. However, its use by the oral route in children younger than 2 years of age is limited due to a lack of pharmacokinetic data. The objectives of the present study were to determine the typical pharmacokinetics of an oral suspension of acyclovir given to children younger than 2 years of age and the interindividual variabilities in the values of the pharmacokinetic parameters in order to support the proposed dosing regimen (24 mg/kg of body weight three times a day for patients younger than 1 month of age or four times a day otherwise). Children younger than age 2 years with HSV or VZV infections were enrolled in a multicenter study. Children were treated for at least 5 days with an acyclovir oral suspension. Plasma samples were obtained at steady state, before acyclovir administration, and at 2, 3, 5, and 8 h after acyclovir administration. Acyclovir concentrations were measured by radioimmunoassay. The data were analyzed by a population approach. Data for 79 children were considered in the pharmacokinetic study (212 samples, 1 to 5 samples per patient). Acyclovir clearance was related to the estimated glomerular filtration rate, body surface area, and serum creatinine level. The volume of distribution was related to body weight. The elimination half-life decreased sharply during the first month after birth, from 10 to 15 h to 2.5 h. Bioavailability was 0.12. The interindividual variability was less pronounced when the parameters were normalized with respect to body weight. Hence, dosage adjustment by body weight is recommended for this population. Simulations showed that the length of time that acyclovir remains above the 50% inhibitory concentration during a 24-h period was more than 12 h for HSV but not for VZV. The proposed dosing regimen seems adequate for the treatment of HSV infections, while for the treatment of VZV infections, a twofold increase in the dose seems necessary for children older than age 3 months.

Pharmacokinetics of netivudine, a potent anti-varicella zoster virus drug, in patients with renal impairment.

The pharmacokinetics of a single oral 200 mg dose of netivudine (1-(beta-D-arabinofuranosyl)-5-(1-propynyl)uracil), a nucleoside analogue under development for use in varicella zoster virus infections, were studied in 12 renal failure (RF) subjects (creatinine clearance 15 +/- 7 mL/min) and 12 age-matched healthy subjects with normal creatinine clearance. Blood and urine samples were collected up to nine days after drug administration. Concentrations of netivudine and of its main metabolite, the pyrimidine base 5-(1-propynyl)uracil (5 PU), were determined by a specific high performance liquid chromatography assay. The mean peak plasma concentrations of netivudine, Tmax, and volume of distribution were not significantly affected by RF. The elimination half-life of netivudine was approximately 15 h in subjects with normal renal function and 60 h in RF patients. Plasma and renal clearances of netivudine were significantly reduced in RF patients and AUC was three to four times higher in these patients. Cmax and AUC of 5 PU were higher in RF patients, and the half-life was also significantly longer. However, the half-life of this metabolite was much lower than that of the parent compound. Tmax and the lag time were similar in the two groups. There were highly significant correlations for netivudine and 5 PU between half-life and creatinine clearance and between renal clearance and creatinine clearance. These findings suggest that netivudine dosage may need to be reduced in patients with severe renal failure, and confirm that formation of the 5 PU is independent of the elimination of netivudine from plasma.

Influence of concurrent antiepileptic medication on the pharmacokinetics of lamotrigine as add-on therapy in epileptic children.

1. Lamotrigine is a new antiepileptic drug, chemically unrelated to currently used antiepileptic medication. Its pharmacokinetics can be influenced by concomitant antiepileptic medication. 2. This study was performed to assess the pharmacokinetic profile of lamotrigine in three groups of children treated with different types of comedication: drugs known to induce, to inhibit or to have no clinically significant influence on drug metabolism, respectively. 3. Thirty-one children aged 6 months to 5 years were included and received a 2 mg kg-1 single oral dose. Lamotrigine plasma profiles were different between the three comedication groups. The half-lives (mean +/- s.d.) were: 7.7 +/- 1.8 h, 21.9 +/- 6.8 h, 44.7 +/- 10.2 h in the "inducer', "other' and "inhibitor' groups respectively. 4. Patients were then dosed to steady state, with the dosage adjusted on the basis of the single dose pharmacokinetics to achieve a minimum plasma concentration between 1.5 and 3 mg l-1. The mean minimum plasma concentration for the three groups was 2.54 +/- 1.28 mg l-1 at steady state. 5. Dosage of lamotrigine can be optimised with knowledge of the metabolic effects of antiepileptic comedication.

Lack of interaction between valaciclovir, the L-valyl ester of acyclovir, and Maalox antacid.

Valaciclovir is rapidly and extensively converted to acyclovir. In this study we investigated the potential interaction between oral valaciclovir and Maalox. On each of three occasions 18 healthy volunteers received a single oral dose of 1000 mg valaciclovir, or 30 mL Maalox 65 min after valaciclovir administration, or 30 mL Maalox 30 min before valaciclovir. Acyclovir plasma concentrations and pharmacokinetic parameters were not significantly affected by administration of Maalox before or after valaciclovir. Therefore, there is no need for restriction of valaciclovir dosing in patients receiving antacid medication.

Pharmacokinetics of lamotrigine in patients with renal impairment: influence of haemodialysis.

The pharmacokinetics of a single 100 mg oral dose of lamotrigine, a new anticonvulsant drug, were studied in six healthy volunteers and in twenty patients with various degrees of renal impairment. Six of these patients were regularly haemodialysed. Lamotrigine concentrations in plasma and urine were measured by the HPLC method. The mean peak plasma concentrations of lamotrigine, tmax, volume of distribution and total clearance were not significantly modified by the degree of renal impairment. The elimination half-life of lamotrigine was approximately 25 h in subjects with normal renal function and 50 h in uraemic patients. These are very large variations in uraemic patients and the results were not statistically significant. Renal clearance of lamotrigine is significantly reduced. Urinary elimination of unchanged and conjugated lamotrigine was reduced in uraemic patients. Thus it seems necessary to treat carefully patients with a very severe renal insufficiency since very large variations in pharmacokinetics were found. A 100 mg oral dose every two days is recommended if creatinine clearance is below 10 ml/min. Haemodialysis shortened the elimination half-life from 59.6 +/- 28.1 h during the interdialysis period to 12.2 +/- 6.4 h during the dialysis period; 17% of the dose was extracted by haemodialysis.

Glucuronidation of 3'-azido-3'-deoxythymidine in human liver microsomes: enzyme inhibition by drugs and steroid hormones.

The molecular form of UDP-glucuronosyltransferase involved in the catalysis of 3'-azido-3'-deoxythymidine (AZT)-5'-O-glucuronide was characterized in human liver microsomes. The specific activity (1.3 nmol/min per mg protein) in transplantable liver was more than 2-times higher than in post-mortem fragments. Liver microsomes from patients suffering Crigler-Najjar syndrome, who are genetically deficient in bilirubin UDP-glucuronosyltransferase, could also glucuronidate AZT to a similar extent, thus indicating that this protein was not involved in that process. A genetically engineered V79 cell line stably expressing a cDNA which encodes a human isozyme active towards 1-naphthol was unable to glucuronidate AZT. Clinically used drugs, most of them being glucuronidated, were tested as potential inhibitors of the glucuronidation of AZT in human liver microsomes. The drugs chemically related to 2-phenylpropionic acid, naproxen and flurbiprofen, and the steroid compounds testosterone, estrone and ethynylestradiol strongly inhibited AZT glucuronidation. Codeine and morphine also decreased the reaction rate although to a lower extent. Except estrone which elicited a partial competitive inhibition, ethynylestradiol, flurbiprofen naproxen and testosterone could competitively inhibit AZT glucuronidation with an apparent Ki of 38, 50, 172 and 250 microM, respectively. The results suggest that these drugs were substrates of the same isozyme(s) involved in AZT glucuronidation. Probenecid was a weak inhibitor of the reaction (Ki 900 microM), only when non-disrupted microsomes were used. This drug may compete with the anion carrier system involved in the microsomal uptake of UDP-glucuronic acid.

In vitro N-glucuronidation of a novel antiepileptic drug, lamotrigine, by human liver microsomes.

The metabolism of the antiepileptic drug lamotrigine was characterized in human liver microsomes. For that purpose a high performance liquid chromatography method allowing the separation of lamotrigine glucuronide from the parent compound, and the quantitation of the glucuronide, was developed. The drug undergoes glucuronidation on the 2-nitrogen atom of the triazine ring, leading to a quaternary ammonium-linked glucuronide. This metabolite was positively identified from its hydrolysis by beta-glucuronidase and its associated radioactivity when UDP-[U-14C] glucuronic acid was used as the cosubstrate. Structural confirmation of the glucuronide was finally obtained by high performance liquid chromatography-mass spectrometry, by using a thermospray interface. The reaction proceeded with an apparent Vmax of 0.65 nmol/min/mg and Km of 2.56 mM. The average value of lamotrigine glucuronidation in four human samples of transplantable liver was 0.43 +/- 0.14 nmol/min/mg, thus indicating a large interindividual variation. An interspecies comparison of hepatic lamotrigine glucuronidation (human, rabbit, rat, monkey) revealed that the rate of glucuronidation was low. Of all the species considered, humans glucuronidated the drug to the greatest extent, with a specific activity 2-fold higher than that observed in rabbit liver microsomes. In contrast, the activity was greater than 20 times lower in monkey (0.019 nmol/min/mg) and at the limit of detection in rat liver microsomes. However, in this species, phenobarbital treatment enhanced lamotrigine glucuronidation slightly (0.017 nmol/min/mg). Among the drugs that undergo quaternary ammonium-linked glucuronidation, chlorpromazine, but not imipramine, amitriptyline and cyproheptadine, inhibited the glucuronidation of lamotrigine in vitro (IC50 of 5.0 x 10(-4) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Immobilization of microsomes into alginate beads is a convenient method for producing glucuronides from drugs.

The production of glucuronides from drugs by immobilized microsomal uridine diphosphate (UDP)-glucuronosyltransferase has been investigated. Of all the immobilization methods used (covalent binding, adsorption by ionic or hydrophobic interactions), only entrapment of microsomes into alginate beads in the presence of polyethyleneimine was effective in producing high glucuronidation rates, thus leading to the formation of large amounts of metabolites. The performance of the bioreactor was optimized with the drug 3'-azido-3'-deoxythymidine (AZT), active against the human immunodeficiency virus, as a model substrate of UDP-glucuronosyltransferase. Calcium (12 mM) could optimally improve the stability of microsomes entrapped in alginate beads. Upon immobilization, enzyme activation occurred, leading to a fivefold increase in specific activity. The determination of apparent Km and Vmax revealed that AZT was a better substrate for the immobilized enzyme than free microsomes. The AZT-glucuronide production obtained after 6 h was threefold higher than that observed with free microsomes. This bioreactor was also efficient in production of glucuronides from structurally different compounds such as bilirubin, 4-nitrophenol, clofibric acid, pirprofen, dextrorphan or morphine, the corresponding glucuronide of which possesses pharmacological or toxicological interest.

Comparison of plasma concentrations of aerosolized pentamidine in nonventilated and ventilated patients with pneumocystosis.

Pentamidine concentrations were determined in plasma after a single aerosolization of 4 mg/kg pentamidine base on 18 patients breathing spontaneously (Group I) and in eight patients receiving mechanical ventilation (Group II). All the patients had documented pneumocystosis. Large interindividual variations in concentrations appeared, especially in Group I. Low concentrations were observed in Group I: Cmax = 65.6 +/- 9.4 micrograms/L (mean +/- SEM), contrasting with high levels in Group II: Cmax = 215.8 +/- 49.8 micrograms/L (mean +/- SEM). Consequently, the mean area under the curve from zero to 4 h was 2.6-fold higher in Group II than in Group I. These findings underline the risk of dose-related pentamidine toxicity in ventilated patients treated with aerosolized pentamidine and the interest of plasma pentamidine monitoring.