Unraveling the drug distribution in brain enabled by MALDI MS imaging with laser-assisted chemical transfer

Accurate localization of central nervous system (CNS) drug distribution in the brain is quite challenging to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), owing to the ionization competition/suppression of highly abundant endogenous biomolecules and MALDI matrix. Herein, we developed a highly efficient sample preparation technique, laser-assisted chemical transfer (LACT), to enhance the detection sensitivity of CNS drugs in brain tissues. A focused diode laser source transilluminated the tissue slide coated with α-cyano-4-hydroxycinnamic acid, an optimal matrix to highly absorb the laser radiation at 405 nm, and a very thin-layer chemical film mainly containing drug molecule was transferred to the acceptor glass slide. Subsequently, MALDI MSI was performed on the chemical film without additional sample treatment. One major advantage of LACT is to minimize ionization competition/suppression from the tissue itself by removing abundant endogenous lipid and protein components. The superior performance of LACT led to the successful visualization of regional distribution patterns of 16 CNS drugs in the mouse brain. Furthermore, the dynamic spatial changes of risperidone and its metabolite were visualized over a 24-h period. Also, the brain-to-plasma (B/P) ratio could be obtained according to MALDI MSI results, providing an alternative means to assess brain penetration in drug discovery.     

Zona thinning with the use of laser: a new approach to assisted hatching in humans

From 1993 to 1994, in our centre, laser-assisted hatching wasperformed on 2- to 4-cell stage embryos obtained from in-vitrofertilization (TVF) patients. We treated 376 embryos from 96patients with repeated IVF failures (two to four attempts) (groupA) and 397 embryos from 111 patients undergoing IVF for thefirst time (group B). Embryos were transferred immediately afterthe laser treatment. Both groups were compared to control groups(A' and B') whose embryos were transferred with intact zonapellucida (ZP). The resulting clinical pregnancies were 41 inA and 44 in B versus 24 hi A' and 23 in B' respectively. Thepregnancy rates per patient were 42.7 and 39.6% versus 23.1and 19% in the control groups (P < 0.05), while the implantationrates per embryo were 12.2% in A and 11.8% in B versus 13 and7.1%. These results show that laser zona thinning of human embryosat 48 h after egg retrieval significantly increases the implantationand pregnancy rate (P < 0.05).     

Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in-vitro fertilization and understanding implantation

It has been shown recently that delayed transfers improve implantationrates in assisted reproductive technology programmes. In a prospectivestudy, the pregnancy rates and safety of outcome were evaluatedin a group of patients after the transfer of day 5 blastocystswith enzymatic treatment of the zona pellucida. Nineteen womenwith a mean age of 32.6 ± 5.2 years and mean 2.1 ±2.2 repeated attempts had blastocyst transfers with a mean numberof 2.5 ± 0.7 embryos replaced per patient. The clinicalpregnancy rates per cycle/transfer and implantation rate were53% and 33%, respectively. The multiple pregnancy rate was 40%(two pregnancies were triplets). The pregnancy and implantationrates were very much higher than observed for most assistedreproduction technology centres. The ‘in-vitro implantation’rates of zona-free blastocysts on a variety of feeder monolayerswas 92%, offering some thoughts as to the role of the zona andinteraction of the inner cell mass and trophoectoderm with theendometrium in implantation. Based on the in-vitro studies andthe high multiple pregnancy rates, it appears that zona-manipulatedblastocysts implant relatively well and there would be a needto reduce the number of transferred embryos to one or two, thusreducing multiple pregnancies and having spare blastocysts availablefor cryopreservation. The results also suggest that using theembryo culture protocol and method of transfer in the presentstudy offers encouraging improvements to assisted reproductiontechnology, and enzymatic treatment of the zona may allow betteranchorage and dialogue of the embryo with the endometrium, helpingus to improve and understand implantation.     

In vitro fertilization in women age 40 and older: The impact of assisted hatching

Purpose To assess the impact of assisted hatching on in vitro fertilization (IVF) outcome in women age 40 and older.Methods A retrospective analysis was performed to compare 28 cycles of IVF without assisted hatching to 38 cycles of IVF with assisted hatching. All patients in both groups were age 40 or older and the mean age was similar.Results The delivery rate per oocyte retrieval was significantly higher in the assisted hatching group (18/38; 48%) compared to the nonhatched controls (3/28; 11%, P=0.0003). The implantation rate of hatched embryos (40/175; 22%) was clearly enhanced, compared to the non-hatched embryos (7/126; 6%, P<0.001). The fertilization rate, number of oocytes and the number of embryos per patient were comparable in the two groups.Conclusions Assisted hatching dramatically improves embryonic implantation and term pregnancy rates in women age 40 and older undergoing IVF.Presented at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, April 3–7, 1995, Vienna, Austria.     

去除人类冷冻胚胎中坏死细胞对其移植结局的影响

目的:评价人类冷冻胚胎解冻后,在胚胎移植前,采用激光辅助孵化及吸出胚胎的坏死细胞碎片是否会提高胚胎的种植率。方法:回顾性的分析了91个冷冻胚胎移植周期的临床妊娠率和胚胎植入率,其中4 1个周期(A组)在胚胎移植前,采用激光辅助孵化结合显微操作去除冷冻胚胎中的坏死细胞碎片,5 0个周期(B组) ,采用激光辅助孵化,但没有去除坏死细胞碎片。采用慢速冷冻 快速解冻的方法。结果:在冷冻胚胎移植前去除胚胎的坏死细胞碎片,能显著性地提高移植周期的临床妊娠率(43.91%vs.2 4 .0 0 % ,P <0 .0 5 )和胚胎植入率(19.4 5 %vs.10 30 % ,P <0 .0 5 )。结论:在人类冷冻胚胎移植前,采用激光及显微操作辅助去除冷冻胚胎的坏死细胞碎片,能有效提高胚胎的种植率。     

A comparison of four different techniques of assisted hatching

BACKGROUND: Assisted hatching (AH) has been proposed as a means to increase the implantation rate in patients with poor prognosis for pregnancy. The procedure appears to be effective when used selectively. Several different methods for AH have been introduced over the years but comparative studies are lacking. The aim of the current study was to compare retrospectively the efficacy of AH performed with four different methods in patients undergoing IVF or ICSI. METHODS: AH was performed prior to day 3 embryo transfer in 794 IVF/ICSI cycles. Indications for AH were females aged >35 years and/or elevated follicular phase FSH levels, previous failed IVF/ICSI cycles, poor embryo quality, and thick zona pellucida (>15 microm). Assignment to one of the four methods of AH was according to the availability of the particular method during the study period. The study was not randomized. RESULTS: Partial zona dissection was used in 239, acid Tyrode in 191, diode laser in 219 and pronase thinning of the zona pellucida in 145. Mean female age, mean number of previous failed IVF/ICSI cycles, number of oocytes retrieved, fertilization and cleavage rates, good quality embryos and zona thickness on day 3 did not differ between groups. Mean number of embryos transferred, implantation rate, clinical pregnancy rate, and abortion rates were likewise similar. CONCLUSIONS: Selective AH using four different methods yields similar implantation and pregnancy rates.