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It is well-established now that position emission tomography (PET) with 2-[18F]fluoro-2-deoxy-glucose (FDG) is a common imaging procedure for mapping cellular glucose metabolism. The uptake of FDG in tumors with higher rates of glycolysis leads to high signal to noise ratios. Despite the tremendous promise of using FDG-PET to detect and monitor tumor metabolism, many indolent tumors are FDG-negative; the tumors may have switched their energy source from glucose to glutamine, thus escaping detection. Glutamine is the most abundant amino acid in the human body. It was recognized early (1960) that glutamine plays an important role in tumor growth and proliferation. We report herein two radiolabeled glutamines, namely L-[5-11C]-glutamine and [18F](2S,4R)4-fluoro-glutamine. These two radioactive glutamines might be useful to map glutamine metabolism in vivo in tumor tissue. We recently developed an efficient method to make L-[5-11C]-glutamine and we could show that L-[5-11C]-glutamine is taken into tumor cells (9L and SF188) and incorporated into protein. Small animal imaging studies on F344 rats bearing 9L xenographed tumors confirmed uptake of the tracer into tumor. But with a short half-life of only 20 minutes, it requires a cyclotron nearby and thus hindering its widespread application. We therefore developed a [18F] fluorinated version (half life of 18F is 110 min)of glutamine, namely [18F](2S,4R)4-fluoro-glutamine. This tracer can be produced and distributed centralized, making it more suitable for clinical use. [18F](2S,4R)4-fluoro-glutamine displayed high cell uptake in 9L and SF188 (derived from human glioblastoma) cell lines and is incorporated into tumor protein. It appears that the transport mechanism may be predominantly through system ASC and may prefer its subtype ASCT2. Small animal imaging studies in F344 rats bearing 9L xenographed tumors, showed rapid uptake into tumor (maximum was reached at 20 min post injection) and the uptake remained constant during the imaging study. The in vivo imaging studies may provide an interesting tool for measuring glutamine metabolism and monitor changes of tumor metabolism.

Within the UK it is illegal to sell or purchase human eggs. Egg donation is permissible and donors may receive a nominal sum as defined by the Human Fertilisation and Embryology Authority. However, the Authority allows egg sharing. The concept and practice of egg trading offends the principle with respect to the trade in human cells, organs and tissues. From April 2005, children conceived with donated gametes will have the right to seek identifying information about the gamete provider when they reach the age of 18 years.

O-(2-[(18)F]fluoroethyl)-L-tyrosine (FET, [(18)F]1) is a useful amino-acid-based imaging agent for brain tumors. This paper reports the synthesis and evaluation of three FET prodrugs, O-(2-[(18)F]fluoroethyl)-L-tyrosyl-L-glycine (FET-Gly, [(18)F]2), O-(2-[(18)F]fluoroethyl)-L-tyrosyl-L-alanine (FET-Ala, [(18)F]3) and N-acetyl O-(2-[(18)F]fluoroethyl)-L-tyrosine (AcFET, [(18)F]4), which could be readily hydrolyzed to FET in vivo for tumor imaging. We investigated their metabolism in the blood and imaging properties in comparison to FET ([(18)F]1).Three new [(18)F]FET derivatives, 2-4, were prepared from their corresponding tosylate-precursors through nucleophilic fluorination and subsequent deprotection reactions. In vitro uptake studies were carried out in 9L glioma cancer cell lines. In vitro and in vivo hydrolysis studies were conducted to evaluate the hydrolysis of FET prodrugs in blood and in Fisher 344 rats. Biodistribution and PET imaging studies were then performed in rats bearing 9L tumors.New FET prodrugs were prepared with 3-28% decay corrected radiochemical yields, good enantiomeric purity (95%) and high radiochemical purity (95%). FET-Gly ([(18)F]2), FET-Ala ([(18)F]3), and AcFET ([(18)F]4) exhibited negligible uptake in comparison to the high uptake of FET ([(18)F]1) in 9L cells. Metabolism studies of FET-Gly ([(18)F]2), FET-Ala ([(18)F]3), and AcFET ([(18)F]4) in rat and human blood showed that FET-Ala ([(18)F]3) was hydrolyzed to FET ([(18)F]1) faster than FET-Gly ([(18)F]2) or AcFET ([(18)F]4). Most of the FET-Ala (79%) was converted to FET ([(18)F]1) within 5min in blood in vivo. Biodistribution studies demonstrated that FET-Ala ([(18)F]3) displayed the highest tumor uptake. The tumor-to-background ratios of FET-Ala ([(18)F]3) and FET ([(18)F]1) were comparable and appeared to be better than those of FET-Gly ([(18)F]2) and AcFET ([(18)F]4). PET imaging studies showed that both FET ([(18)F]1) and FET-Ala ([(18)F]3) could visualize tumors effectively, and that they share similar imaging characteristics.FET-Ala ([(18)F]3) demonstrated promising properties as a prodrug of FET ([(18)F]1), which could be used in PET imaging of tumor amino acid metabolism.

The purpose of the present study was (i) to assess the value of using a low dose of hMG (75 IU/day) to achieve ovarian stimulation in women who have previously shown an exaggerated response to a standard dose of 150 IU human menopausal gonadotropin/day in a desensitization (group I) or flare-up (group II) protocol and (ii) to determine whether the choice of GnRH-a regimen in a subsequent cycle, namely, a desensitization or flare-up protocol, influenced the effectiveness of the low dose of hMG.In group I, 75% (12/16) and 57% (8/14) of the subsequent desensitization and flare-up protocols, respectively, were cancelled because of inadequate ovarian response. Similarly, the cancellation rates in group II were 10 of 10 and 7 of 11 (64%), respectively. The total cancellation rate (groups I and II together) with the desensitization protocol was higher than that using the flare-up protocol (P0.05).The simple use of a reduced dose of hMG (75 IU/day) for subsequent in vitro fertilization in women to minimize the risk of the development of ovarian hyperstimulation is of limited benefit since a large proportion then shows an inadequate response. This is particularly pronounced with a subsequent desensitization protocol which does not utilize endogenous gonadotropins to initiate follicular development.

Objectives: The nuclear enzyme PARP-1 plays a central role in sensing DNA damage and facilitating repair. Tumors with BRCA1/2 mutations are highly dependent on PARP-1 as an alternative mechanism for DNA repair, and PARP inhibitors generate synthetic lethality in tumors with BRCA mutations, resulting in cell cycle arrest and apoptosis. Zhou et al. recently synthesized an 18F-labeled PARP-1 inhibitor (18F-FluorThanatrace) for PET, and demonstrated high specific tracer uptake in a xenograft model of breast cancer (Zhou, Bioorg Med Chem, 22:1700, 2014). The current study seeks to quantify the relationship between 18F-FluorThanatrace binding (both in vitro and on PET imaging of human tumor xenografts) and the level of constitutively active PARP-1, using multiple human breast cancer cell lines, including a BRCA1 defective line. Methods: BRCA1 defective HCC1937, triple negative MDA-MB-231, and luminal A MCF-7 human breast cancer lines were assessed for constitutive PARP-1 activity via a chemiluminescent ELISA assay for PAR and by Western blot. The same cell lines were incubated with 18F-FluorThanatrace over various time increments, and tracer uptake was assayed via a gamma counter. Specificity of tracer binding was verified via co-incubation with competitive inhibitor Olaparib, and specific tracer uptake was calculated as the difference between uptake with and without Olaparib. Specific tracer uptake was compared to levels of constitutive PARP-1 activity in all cell lines. In addition, HCC1937 and MDA-MB-231 xenograft tumor models were imaged via 18F-FluorThanatrace-PET/CT, and PET uptake was correlated with PARP-1 activity. Results: BRCA1-defective HCC1937 had higher constitutive PARP-1 activity than cell lines with intact BRCA1. In vitro levels of 18F-FluorThanatrace uptake correlated with constitutive PARP-1 activity across cell lines. In addition, 18F-FluorThanatrace measured by PET in xenograft breast cancer tumor models correlated with constitutive PARP-1 activity. Conclusions: Tumor uptake of 18F-FluorThanatrace, both in vitro and on PET imaging of xenograft tumor models, quantitatively reflects differences in PARP-1 activity across different breast cancer cell lines, including BRCA1 defective. This motivates further studies of 18F-FluorThanatrace as an in vivo measure of PARP-1 activity and possibly as a predictive marker for PARP-1 therapy in patients, including those with BRCA1/2 mutations. Citation Format: Edmonds CE, Lieberman BP, Xu K, Zeng C, Makvandi M, Li S, Hou C, Lee H, Greenberg RA, Mankoff DA, Mach RH. 18F-radiolabeled PARP-1 inhibitor uptake as a marker of PARP-1 activity in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-01-06.

Introduction: Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer and typically results in poor patient prognosis. While other breast cancer subtypes benefit from targeted therapies, TNBC currently has no approved agents. The sigma-2 receptor has been validated as a proliferation marker and also a site for therapeutic drug delivery in ovarian and pancreatic cancer. In this study we evaluated the sigma-2 receptor as a target for therapeutic drug delivery in TNBC using a sigma-2 targeting vehicle conjugated to a small molecule activator of caspases(SMAC) mimetic, SW-IV-134. Methods: Triple negative breast cancer cell lines; MDA-MB-231, HCC1937 and HCC1806 were tested for sensitivity to sigma-2 targeted compound SW-IV-134 vs. Taxol or the deconstructed conjugate subparts. 2 hr and 48 hr treatments we assessed to determine whether there was a significant in vitro targeting effect. Sigma-2 expression was quantified through radioligand saturation studies using cell homogenate tissue and the Ki value of SW-IV-134 for the sigma-2 receptor was calculated through competitive inhibition studies. Caspase 3/7 activation was assessed after 2 or 48 hr treatments. Results: SW-IV-134 was the most potent treatment in MDA-MB-231 and HCC1937, while no difference from taxol was found in HCC1806. Sigma-2 receptor expression was found to be the highest in the MDA-MB-231 and no difference was found in HCC1937 and HCC1806. The Ki of SW-IV-134 for the sigma-2 receptor was 23 nM and is consistent with previously reported values. Caspase 3/7 activation was detected in all cell lines following treatment at 2 and 48 hr. The greatest caspase 3/7 activation was observed in the MDA-MB-231 cell line. Conclusion: The sigma-2 receptor is expressed at different levels on the three TNBC cell lines and the receptor expression corresponds to therapeutic efficacy. SW-IV-134 was the most potent therapeutic tested in two of the three cell lines with no difference between taxol observed in HCC1806. This study shows the sigma-2 receptor is a target that can be exploited for therapeutic drug delivery in three TNBC cell lines and may provide a platform for future targeted therapy development for TNBC. Citation Format: Mehran Makvandi, Estifanos D. Tilahun, Brian P. Lieberman, Kuiying Xu, Chenbo Zeng, Catherine Hou, Robert H. Mach. The sigma-2 receptor as a target for therapeutic drug delivery in triple negative breast cancer. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B42.

In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) is an effective treatment for severe male factor infertility, but the expertise and equipment required for ICSI limit the provision of this service in smaller IVF centers. Kingsland et al. (1) introduced the concept of transport IVF in the United Kingdom. In a transport IVF program, the couple is managed clinically at the peripheral unit. The egg recovery is undertaken at the peripheral hospital. The IVF and ICSI are performed at the central unit. The embryos usually are replaced at the central unit (2). The Wirral Hospital is situated 40 miles from Manchester, United Kingdom. The unit has facilities for IVF but not ICSI. From 1996 to 1998 (period A), both the egg recoveries and embryo replacements were undertaken at the peripheral unit, and the ICSI was performed at the central laboratory at St. Mary’s Hospital, Manchester. Thus transport of oocytes to the central unit and embryos back to the peripheral unit was necessary. During this time, the central unit performed ICSI for two other peripheral units, for which process the eggs were also recovered at the peripheral unit, but the ICSI and embryo replacement were performed at the central unit, that is, embryos were not transported. The policy with respect to Wirral Hospital was revised in 1999 because of very poor results as compared with the other peripheral units; since then (period B), the ICSI and the replacements have been performed at the central unit to eliminate transport of embryos.

(18)F-labeled (2S,4R)-4-fluoro-l-glutamine (4F-GLN) has demonstrated high uptake in tumor cells that undergo high growth and proliferation. Similar tumor targeting properties have also been observed for (18)F-labeled (2S,4R)-4-fluoro-l-glutamate (4F-GLU), suggesting that both are useful imaging agents. A new labeling procedure facilitates the preparation of (18)F-(2S,4R)4F-GLN and (18)F-(2S,4R)4F-GLU with confirmed radiochemical and enantiomeric purity. Here, we report the preparation and comparative evaluation of (18)F-(2S,4R)4F-GLN and (18)F-(2S,4R)4F-GLU as tumor metabolic imaging agents.Uptake of enantiomerically pure (18)F-(2S,4R)4F-GLN and (18)F-(2S,4R)4F-GLU was determined in 3 tumor cell lines (9L, SF188, and PC-3) at selected time points. The in vitro cell uptake mechanism was evaluated by inhibition studies in 9L cells. In vivo biodistribution and PET studies were performed on male F344 rats bearing 9L tumor xenografts.In vitro cell uptake studies showed that (18)F-(2S,4R)4F-GLN displayed higher uptake than (18)F-(2S,4R)4F-GLU. Amino acid transport system ASC (alanine-serine-cysteine-preferring; in particular, its subtype ASCT2 [SLC1A5 gene]) and system X(c)(-) (SLC7A11 gene) played an important role in transporting (18)F-(2S,4R)4F-GLN and (18)F-(2S,4R)4F-GLU, respectively, across the membrane. After being transported into cells, a large percentage of (18)F-(2S,4R)4F-GLN was incorporated into protein, whereas (18)F-(2S,4R)4F-GLU mainly remained as the free amino acid in its original form. In vivo studies of (18)F-(2S,4R)4F-GLN in the 9L tumor model showed a higher tumor uptake than (18)F-(2S,4R)4F-GLU, whereas (18)F-(2S,4R)4F-GLU had a slightly higher tumor-to-background ratio than (18)F-(2S,4R)4F-GLN. Imaging studies showed that both tracers had fast accumulation in 9L tumors. Compared with (18)F-(2S,4R)4F-GLU, (18)F-(2S,4R)4F-GLN exhibited prolonged tumor retention reflecting its incorporation into intracellular macromolecules.Differences in uptake and metabolism in tumor cells were found between (18)F-(2S,4R)4F-GLN and (18)F-(2S,4R)4F-GLU. Both agents are potentially useful as metabolic tracers for tumor imaging.

A novel series of tropane derivatives containing a fluorinated tertiary amino or amide at the 2β position was synthesized, labeled with the positron-emitter fluorine-18 (t(1/2)=109.8 min), and tested as potential in vivo dopamine transporter (DAT) imaging agents. The corresponding chlorinated analogs were prepared and employed as precursors for radiolabeling leading to the fluorine-18-labeled derivatives via a one-step nucleophilic aliphatic substitution reaction. In vitro binding results showed that the 2β-amino compounds 6b, 6d and 7b displayed moderately high affinities to DAT (K(i)10nM). Biodistribution studies of [(18)F]6b and [(18)F]6d showed that the brain uptakes in rats were low. This is likely due to their low lipophilicities. Further structural modifications of these tropane derivatives will be needed to improve their in vivo properties as DAT imaging agents.

Objectives Recently, 9-[ 18 F]fluoropropyl-(+)-dihydrotetrabenazine ( 18 F-AV-133) was reported as a new vesicular monoamine transporter (VMAT2) imaging agent for diagnosis of Parkinson's disease (PD). To shorten the preparation of 18 F-AV-133 and to make it more widely available, we evaluated a simple, rapid purification with a solid-phase extraction method (SPE) using an Oasis HLB cartridge instead of high pressure liquid chromatography (HPLC). The SPE method produced doses containing a pseudo-carrier, 9-hydroxypropyl-(+)-dihydrotetrabenazine (AV-149). Methods To test the possible side effects of this pseudo-carrier, comparative dynamic PET scans of the brains of normal monkeys (2 each) and uni-laterally 6-OH-dopamine-lesioned PD monkeys (2 each) were performed using 18 F-AV-133 doses prepared by either SPE (containing pseudo-carrier) or HPLC (containing no pseudo-carrier). Autoradiographs of post mortem monkey brain sections were evaluated to confirm the relative 18 F-AV-133 uptake in the PD monkey brains and the effects of the pseudo-carrier on VMAT2 binding. Results The radiochemical purity of the 18 F-AV-133, whether prepared by SPE or by HPLC, was excellent (>99%). PET scans of normal and PD monkey brains showed an expected reduction of VMAT2 in the lesioned areas of the striatum. It was not affected by the presence of the pseudo-carrier, AV-149 (maximally 250μg/dose). The reduced uptake in the striatum of the lesioned monkey brains was confirmed by autoradiography. Ex vivo inhibition studies of 18 F-AV-133 binding in rat brains, conducted with increasing amounts of AV-149, suggested that at the highest concentration (3.5mg/kg) the VMAT2 binding in the striatum was only moderately blocked (20% reduction). Conclusions The pseudo-carrier, AV-149, did not affect the 18 F-AV-133/PET imaging of VMAT2 binding sites in normal or uni-laterally lesioned monkey brains. The new streamlined SPE purification method will enable 18 F-AV-133 to be widely available for routine clinical application in determining changes in monoamine neurons for patient with movement disorders or other psychiatric illnesses.

The Human Fertilisation and Embryology Act became law in the UK in 1990, and a statutory body, the Human Fertilisation and Embryology Authority, was established to administer the Act. The opinions of those persons responsible for licensed activity under the Act were canvassed anonymously to assess the initial effect of the Act on their activities, and the administration of the Act by the 'Authority'. The views expressed reflect the opinions of 80% of the 'responsible persons' and are thus likely to be of value to those responsible for administration of the Act and also those planning legislation in this field of human endeavour.

Changes in gene expression, metabolism, and energy requirements are hallmarks of cancer growth and self-sufficiency. Upregulation of the PI3K/Akt/mTor pathway in tumor cells has been shown to stimulate aerobic glycolysis, which has enabled 18F-FDG PET tumor imaging. However, of the millions of 18F-FDG PET scans conducted per year, a significant number of malignant tumors are 18F-FDG PET–negative. Recent studies suggest that several tumors may use glutamine as the key nutrient for survival. As an alternative metabolic tracer for tumors, 18F-(2S,4R)4-fluoroglutamine was developed as a PET tracer for mapping glutaminolytic tumors. Methods: A series of in vitro cell uptake and in vivo animal studies were performed to demonstrate tumor cell addiction to glutamine. Cell uptake studies of this tracer were performed in SF188 and 9L glioblastoma tumor cells. Dynamic small-animal PET studies of 18F-(2S,4R)4-fluoroglutamine were conducted in 2 animal models: xenografts produced in F344 rats by subcutaneous injection of 9L tumor cells and transgenic mice with M/tomND spontaneous mammary gland tumors. Results: In vitro studies showed that both transformed 9L and SF188 tumor cells displayed a high rate of glutamine uptake (maximum uptake, ≈16% dose/100 μg of protein). The cell uptake of 18F-(2S,4R)4-fluoroglutamine by SF188 cells is comparable to that of 3H-l-glutamine but higher than that of 18F-FDG. The tumor cell uptake can be selectively blocked. Biodistribution and PET studies showed that 18F-(2S,4R)4-fluoroglutamine localized in tumors with a higher uptake than in surrounding muscle and liver tissues. Data suggest that certain tumor cells may use glutamine for energy production. Conclusion: The results support that 18F-(2S,4R)4-fluoroglutamine is selectively taken up and trapped by tumor cells. It may be useful as a novel metabolic tracer for tumor imaging.

β-Amyloid plaques (Aβ plaques) in the brain are associated with cerebral amyloid angiopathy (CAA). Imaging agents that could target the Aβ plaques in the living human brain would be potentially valuable as biomarkers in patients with CAA. A new series of (18)F styrylpyridine derivatives with high molecular weights for selectively targeting Aβ plaques in the blood vessels of the brain but excluded from the brain parenchyma is reported. The styrylpyridine derivatives, 8a-c, display high binding affinities and specificity to Aβ plaques (K(i) = 2.87, 3.24, and 7.71 nM, respectively). In vitro autoradiography of [(18)F]8a shows labeling of β-amyloid plaques associated with blood vessel walls in human brain sections of subjects with CAA and also in the tissue of AD brain sections. The results suggest that [(18)F]8a may be a useful PET imaging agent for selectively detecting Aβ plaques associated with cerebral vessels in the living human brain.

A series of novel N -fluoropyridyl-containing tropane derivatives were synthesized and their binding affinities for the dopamine transporter (DAT), serotonin transporter (SERT) and norepinephrine (NET) were determined via competitive radioligand binding assays. Among these derivatives, compound 6d showed the highest binding affinity to DAT ( K i = 4.1 nM), and selectivity for DAT over SERT (5-fold) and NET (16-fold). Compound 6d was radiolabeled with Fluorine-18 in two steps. Regional brain distribution and ex vivo autoradiography studies of [ 18 F] 6d demonstrated that the ligand was selectively localized in the striatum region, where DAT binding sites are highly expressed. [ 18 F] 6d may be useful as a potential radioligand for imaging DATs with PET.

Rationale and Objectives Recent reports suggest that cancer cells may use glutamine, instead of glucose, as an alternative source of metabolic energy. This suggests that hyperpolarized 13 C glutamine may be useful as a magnetic resonance spectroscopy (MRS) imaging agent for detecting changes in glutamine metabolism in cancerous cells or tissues. Materials and Methods Synthesis of [5- 13 C-4- 2 H 2 ]-L-glutamine was accomplished through a seven-step synthetic pathway with a 44% overall yield. The introduction of two stable isotopes was performed by a NaB 2 H 4 -mixed anhydride reduction and K 13 CN-nuclophilic substitution, respectively. The desired [5- 13 C-4- 2 H 2 ]-L-glutamine was successfully obtained by a one-pot reaction of deprotection and controlled cyanide hydrolysis. Hyperpolarized [5- 13 C-4- 2 H 2 ]-L-glutamine samples were tested in human glioma cells (myc upregulated glia cells, SF188-Bcl-x L ). MRS signals were obtained with a 9.4 Tesla 89-mm bore nuclear magnetic resonance spectrometer and a direct-detection multi-nuclear probe. Results The initial degree of polarization for [5- 13 C-4- 2 H 2 ]-L-glutamine was ∼5% and the initial 13 C signal to noise ratio was ∼100:1. Glutamate was detected within seconds after the injection of hyperpolarized glutamine into the cells. The ratio of glutamate to glutamine was very high, indicating rapid conversion to glutamate. Similar cell uptake studies using [ 3 H]-L-glutamine also demonstrated cell uptakes higher than that of [ 18 F]fluorodeoxyglucose. Conclusion We are reporting the first example of using specifically deuterated [5- 13 C-4- 2 H 2 ]-L-glutamine in conjunction with hyperpolarized MRS for studying "glutaminolysis" in proliferating tumor cells.

In the search of new probes for in vivo brain imaging of vesicular monoamine transporter type 2 (VMAT2), we have developed an efficient synthesis of a novel series of 3-alkyl-dihydrotetrabenazine (DTBZ) derivatives. The affinity of VMAT2 was evaluated by an in vitro inhibitory binding assay using [ 125 I]-iodovinyl-TBZ or [ 18 F](+)-FP-DTBZ as radioligands in rat striatal tissue homogenates. New DTBZ derivatives exhibited moderate to good binding affinity to VMAT2. Among these new ligands, compound 4b showed the best affinity for VMAT2 ( K i = 5.98 nM) and may be a useful lead compound for future structure–activity studies.

Testing for antisperm antibodies (ASAs) is an important part of the work-up of the sub-fertile couple, yet there is little consensus regarding the most appropriate methods. The SpermCheck assay (GSC; Bio-Rad Laboratories Inc., Diagnostics Division, Hercules, CA, U.S.A.) is supplied with wash buffer, controls and bead reagent which detects all three major classes of ASAs (IgA, IgG and IgM) in a single test. This study compared results on a bank of samples using the tray agglutination test (TAT), immunobead test (IBT), GSC and a modified SpermCheck assay to detect a single isotype in each test (SISC). The IBT and SISC showed excellent correlation, with 127/141 (90.1%) tests agreeing. There was an apparent lack of sensitivity to IgM with GSC as 8/15 (53.3%) samples testing positive with IBT and 7/15 (46.7%) testing positive with SISC were negative with GSC. Of the 24 IBT-negatives, seven (29.2%) were positive for TAT, indicating a high incidence of non-immunological agglutination, though this decreased as the TAT titre increased. The proportion of samples testing positive for IBT increased with TAT titre: 3/20 (15.0%) for TAT-negative samples, 6/10 (60.0%) for low titres and 21/24 (87.5%) for high titres. This was also observed when comparing the GSC with TAT. The TAT therefore appears useful as a first-line screen, whilst the inability of the GSC to adequately detect IgM limits its use as an indirect test. Both the IBT and SISC can be used to further investigate the type and class of ASA present.

The effects of supplementary growth hormone (GH) treatment upon insulin-like growth factor-1 (IGF-1), IGF binding protein-3 (IGFBP-3) concentrations in serum and ovarian follicular fluid were investigated in women undergoing buserelin human menopausal gonadotrophin (HMG) ovulation induction for in-vitro fertilization. Women (n = 40), aged 24-39 (mean 35 years), who showed poor ovarian responses to HMG, were recruited and randomly divided into two groups. Each patient received two cycles of ovulation induction, one with GH (12 IU/day x 12 days/HMG/buserelin) and another with placebo/HMG. Serum IGF-1 increased substantially during the GH treatment and remained significantly higher than the control 2 days after the last GH injection. Serum IGFBP-3 fell steadily during the placebo/HMG treatment and to a nadir on the day of oocyte retrieval (P < 0.05 compared to serum before any treatment). In contrast, IGFBP-3 was increased (P < 0.01) during the GH administration and returned to the control level 2 days after GH injection. Serum oestradiol concentrations on the eighth day of HMG and the day of human chorionic gonadotrophin (HCG) were not significantly different between the two groups. Serum IGF-1 was highly correlated with IGFBP-3 before any treatment (r = 0.433, P < 0.001). This correlation disappeared after buserelin, placebo/HMG treatment in the control group, but it was maintained during GH/HMG treatment (r = 0.343, P = 0.04). Follicular fluid concentrations of GH and IGF-1, not IGFBP-3 or oestradiol, were significantly elevated in the GH-treated women.(ABSTRACT TRUNCATED AT 250 WORDS)

Objectives To analyse the incidence and factors associated with the ovarian hyper-stimulation syndrome (OHS) in our IVF/GIFT programme before and after the introduction of a strategy to cryopreserve all embryos from women judged to be at risk. Design Two hundred forty-one consecutive IVF/GIFT cycles from January to December 1989. Setting Specialist fertility unit, Manchester, UK. Interventions Pituitary suppression was effected by a daily subcutaneous injection of buserelin (500 μg) beginning 7 days before the expected menses. The ovarian stimulation was with variable amounts of human menopausal gonadotrophin. Ovulation was induced with 10 000 i.u. human chorionic gonadotrophin (hCG). From January to May (period A), gametes/embryos were replaced and 2000 i.u. hCG given, irrespective of the serum oestradiol (E2) concentration. From June to December (period B), all the embryos from women with an E2>3500 pg/ml on the day of ovulatory trigger were electively cryopreserved. Main outcome measures Serum E2, features of moderate or severe OHS, clinical pregnancies. Results The OHS occurred in 10/105 (9.5%) and 12/136 (8.8%) cycles in periods A and B, respectively. Fewer women (6% versus 60%, P 3500 pg/ml who became pregnant after gamete/embryo transfer in period A. The main factors associated with the development of OHS were serum E2 concentrations >3500 pg/ml, whether gamete/embryos were replaced and the additional hCG given, the occurrence of a pregnancy and the presence of polycystic ovary disease. Conclusion The elective cryopreservation of all embryos from women with high E2 levels reduced the severity, but not the incidence of symptomatic OHS.

Two cases are reported of successful pregnancies following long-term semen banking prior to chemotherapy and radiotherapy for malignancy. With the first case, the patient banked semen at the age of 20 years prior to chemotherapy for Hodgkin's disease; 11 years later the thawed semen was used for IVF with intracytoplasmic sperm injection (ICSI), resulting in twins being born following the transfer of frozen-thawed embryos. In the second case, the patient banked semen at the age of 17 years prior to chemotherapy and radiotherapy for acute myeloid leukaemia; 8 years later it was used for ICSI, resulting in triplets being born following the transfer of frozen-thawed embryos. These cases support long-term semen banking for men whose future fertility may be compromised by suppression of spermatogenesis secondary to administration of chemo/radiotherapy treatment. The advent of successful ICSI combined with embryo cryopreservation has increased the chance of thawed cryopreserved semen achieving fertilization. Banking of a single ejaculate prior to commencement of chemotherapy/radiotherapy treatment may preserve potential fertility without compromising the oncology treatment.

IVF treatments carry a high risk of twin pregnancy which confers a higher risk to the mother and child than singletons. Increased use of elective single embryo transfer (eSET) can reduce this twin rate. We aimed to utilize a previously published data set and statistical model based on routinely collected clinical data to predict the outcomes of policies that increase the proportion of eSET.The models allow simultaneous prediction of outcomes from double embryo transfer (DET) and SET. These models were used to predict outcomes for different scenarios using SET in both the initial (fresh) transfer and over a complete cycle (transfer of all embryos created, with cryopreservation). A total of 16 096 cycles (12 487 fresh and 3609 frozen) from 9040 couples treated between 2000 and 2005 were included in the final analyses.For any transfer, SET has about a one-third lower live birth rate relative to DET: this can be partially mitigated by appropriate patient and treatment cycle selection, with several realistic policies performing similarly. However, if we consider complete cycles with embryo cryopreservation, it is possible for repeat SET to produce more live births per egg retrieval than repeat DET.All patients receiving SET would have a higher chance of successful treatment in that cycle if they received DET. The selection of appropriate patients for SET can partially ameliorate the overall loss. For complete cycles, repeat SET could produce more live births per egg retrieval than repeat DET. All treatments involving SET will increase the number of treatments required to achieve a successful outcome and this extra treatment burden will be a significant barrier to the implementation of such treatments.

Introduction l-Amino acid-based tracers have established their important role as tumor metabolic imaging agents. Recently, a number of studies demonstrated that d-amino acids may have improved imaging properties than their corresponding l-isomers. We synthesized and evaluated the d-isomer of a new phenylalanine derivative, p -(2-[ 18 F]fluoroethyl)-phenylalanine ([ 18 F]FEP), in comparison to its l-isomer and previously reported the l- and d-isomers of O -(2-[ 18 F]fluoroethyl)-tyrosine ([ 18 F]FET). Methods l- and d-Isomers of [ 18 F]FET and [ 18 F]FEP were successfully synthesized via a rapid and efficient two-step nucleophilic fluorination and deprotection reaction. In vitro studies were carried out in 9L glioma cells. In in vivo studies, Fisher 344 rats bearing the 9L tumor model were used. Results l- and d-Isomers of 18 F-fluoroalkyl tyrosine and phenylalanine derivatives were efficiently labeled with high enantiomeric purity (>95%), good yield (11–45%) and high specific activity (21–75 GBq/μmol). d-[ 18 F]FEP showed a similar linear time-dependent uptake as d-[ 18 F]FET, while their corresponding l-isomers had much faster and higher uptake (4.3- to 16.0-fold at maximum uptake). The maximum uptake of the new compounds, l- and d-[ 18 F]FEP, was 1.4- and 5.2-fold of that reported for l- and d-[ 18 F]FET, respectively. Transport characterization studies indicated that both l- and d-[ 18 F]FEP were selective substrates for system L. While l-[ 18 F]FEP exhibited preference towards one subtype of system L, LAT1, d-[ 18 F]FEP did not exhibit the same preference. Small animal PET imaging studies showed that both l- and d-[ 18 F]FEP had higher uptake in 9L tumor compared to surrounding tissues, but d-isomer had lower tumor-to-muscle ratio in comparison with its l-isomer. Conclusion Both l- and d-[ 18 F]FEP are substrates for system L amino acid transporter with different preference toward its subtypes. Small animal imaging studies of 9L tumor showed that d-[ 18 F]FEP did not show better imaging properties than their corresponding l-isomer.

BACKGROUND: In vitro fertilisation (IVF) treatments involve an egg retrieval process, fertilisation and culture of the resultant embryos in the laboratory, and the transfer of embryos back to the mother over one or more transfer cycles. The first transfer is usually of fresh embryos and the remainder may be cryopreserved for future frozen cycles. Most commonly in UK practice two embryos are transferred (double embryo transfer, DET). IVF techniques have led to an increase in the number of multiple births, carrying an increased risk of maternal and infant morbidity. The UK Human Fertilisation and Embryology Authority (HFEA) has adopted a multiple birth minimisation strategy. One way of achieving this would be by increased use of single embryo transfer (SET). OBJECTIVES: To collate cohort data from treatment centres and the HFEA; to develop predictive models for live birth and twinning probabilities from fresh and frozen embryo transfers and predict outcomes from treatment scenarios; to understand patients' perspectives and use the modelling results to investigate the acceptability of twin reduction policies. METHODS: A multidisciplinary approach was adopted, combining statistical modelling with qualitative exploration of patients' perspectives: interviews were conducted with 27 couples at various stages of IVF treatment at both UK NHS and private clinics; datasets were collated of over 90,000 patients from the HFEA registry and nearly 9000 patients from five clinics, both over the period 2000-5; models were developed to determine live birth and twin outcomes and predict the outcomes of policies for selecting patients for SET or DET in the fresh cycle following egg retrieval and fertilisation, and the predictions were used in simulations of treatments; two focus groups were convened, one NHS and one web based on a patient organisation's website, to present the results of the statistical analyses and explore potential treatment policies. RESULTS: The statistical analysis revealed no characteristics that specifically predicted multiple birth outcomes beyond those that predicted treatment success. In the fresh transfer following egg retrieval, SET would lead to a reduction of approximately one-third in the live birth probability compared with DET, a result consistent with the limited data from clinical trials. From the population or clinic perspective, selection of patients based on prognostic indicators might mitigate about half of the loss in live births associated with SET in the initial fresh transfer while achieving a twin rate of 10% or less. Data-based simulations suggested that, if all good-quality embryos are replaced over multiple frozen embryo transfers, repeated SET has the potential to produce more live birth events than repeated DET. However, this would depend on optimising cryopreservation procedures. Universal SET could both reduce the number of twin births and lead to more couples having a child, but at an average cost of one more embryo transfer procedure per egg retrieval. The interview and focus group data suggest that, despite the potential to maintain overall success rates, patients would prefer DET: the potential for twins was seen as positive, while additional transfer procedures can be emotionally, physically and financially draining. CONCLUSIONS: For any one transfer, SET has about a one-third loss of success rate relative to DET. This can be only partially mitigated by patient and treatment cycle selection, which may be criticised as unfair as all patients receiving SET will have a lower chance of success than they would with DET. However, considering complete cycles (fresh plus frozen transfers), it is possible for repeat SET to produce more live births than repeat DET. Such a strategy would require support from funders and acceptance by patients of both cryopreservation and the burden of additional transfer cycles. Future work should include development of improved clinical and regulatory database systems, surveys to quantify the extent of patients' beliefs and experiences and develop approaches to meet their information needs, and, ideally, randomised controlled trials comparing policies of repeated SET with repeated DET.

Objective To create developmentally competent embryos from failed-to-fertilize oocytes for use in infertility research and human embryonic stem cell derivation. Design Attempts to recover developmental potential of failed-to-fertilize oocytes were made by using either parthenogenetic activation or reinsemination by intracytoplasmic sperm injection. Resulting embryos were cultured to various stages up to and including blastocyst, and single embryos exhibiting normal development were analyzed for gene expression by quantitatively profiling representative transcripts. Setting Hospital-based assisted reproductive technology laboratory and University academic laboratories. Patient(s) One hundred sixty-five couples undergoing assisted fertility treatment. Intervention(s) Metaphase II stage oocytes were either parthenogenetically activated or reinseminated with donor sperm, then allowed to develop up to and including the blastocyst stage. Main Outcome Measure(s) Gene expression analysis was performed on oocytes and embryos by quantitative reverse transcriptase–polymerase chain reaction for markers of developmental competence. Result(s) Fertilization occurred in 65% of the activated or reinseminated oocytes, which resulted in a blastocyst formation rate of 8%. Evaluation of a number of developmentally important genes in those embryos exhibiting normal development revealed profile and levels of expression similar to control embryos. One blastocyst from an activated oocyte yielded a novel pluripotent stem cell line indistinguishable from those derived from embryos surplus to infertility treatment. Conclusion(s) Clinically unusable oocytes represent a valuable alternative source of normal human embryos for human infertility and stem cell research without conflicting with patient treatment.

Three new 18F labeled fluoroalkyl tyrosine derivatives, O-(2-[18F]fluoroethyl)-alpha-methyltyrosine (FEMT, [18F]2), O-(2-[18F]fluoroethyl)-2-L-azatyrosine (FEAT, [18F]3), O-(2-[18F]fluoroethyl)-L-tyrosineamide (FETA, [18F]4) have been synthesized and radiofluorinated with 5-34% decay-corrected yield. In vitro studies were carried out in U-138 MG human glioblastoma. Cellular uptake of new tracers was compared to clinically utilized imaging agent O-(2-[18F]fluoroethyl)-L-tyrosine (FET, [18F]1). The uptake of tracers followed the order of FET ([18F]1) > FEAT([18F]3) > FEMT ([18F]2) approximately FETA ([18F]4).

From 1st June 1989 to 31st May 1991, 78 women with a serum oestradiol level greater than 3500 pg/ml on the day of the ovulatory trigger, following pituitary suppression with buserelin and ovarian stimulation with human menopausal gonadotrophins (HMG), had all their embryos electively cryopreserved at the pronucleate stage to minimize the risk of developing ovarian hyperstimulation syndrome (OHS). Treatment with buserelin was continued in the luteal phase. A median of 19 oocytes (range 7-43) was obtained and 12 embryos (range 1-37) frozen per cycle. Twenty-one (27%) women developed OHS (six severe). Women developing OHS had higher (P less than 0.05) serum oestradiol concentrations on the 7th day after oocyte retrieval, compared to those who did not. No differences were found for any of the following criteria: aetiology of infertility, age, total dose of HMG, number of oocytes, fertilization rate or freeze-thaw survival of embryos. Subsequently, 125 frozen-thawed embryo replacements have been undertaken, using buserelin and hormone replacement therapy (HRT) (n = 93) or natural cycles (n = 32). The overall freeze-thaw survival and implantation rates per embryo were 71.8 and 11.7%, respectively. The pregnancy rates in natural cycles (19%) and buserelin/HRT cycles (29%) were not significantly different.

Biosynthetic human growth hormone added to an ovarian stimulation regime of human menopausal gonadotrophin (HMG) for IVF treatment improves the response of women who were previously resistant. This study investigated the efficacy of growth hormone (GH)/buserelin/HMG treatment in women with a previous normal response to buserelin/HMG stimulation. Ten patients (28-36 years, mean 32.5 years) were treated with GH (6 IU/day) plus buserelin/HMG. A control group of 10 women (28-37 years mean 31.0 years) received buserelin/HMG alone. All were given buserelin 500 micrograms and 2 ampoules (150 IU) HMG daily once pituitary suppression had been confirmed. There was no improvement in the GH group as assessed by follicular growth rate or number, oocyte number per woman and pregnancy rate. There was no effect of GH upon the serum oestradiol level and the follicular fluid levels of oestradiol, GH and inhibin. Serum IGF-1 increased significantly during GH administration, returning to pre-treatment levels 2 days after the last dose of GH. Follicular IGF-1 was much higher in the GH-treated group than the controls. Significant correlations were found in the GH-treated group between follicular fluid GH and follicular fluid oestradiol concentrations and between follicular GH and follicular size. Follicular IGF-1 was correlated with the serum IGF-1 concentration on day 8 of the GH/HMG treatment. In conclusion GH/buserelin/HMG treatment in women with a previous normal response to buserelin/HMG stimulation increased their serum and follicular IGF-1 concentrations. However, it does not improve the clinical ovarian response or the follicular secretion of oestradiol or inhibin.

Secretory differentiation of endometrium after multiple follicular stimulation using gonadotrophin releasing hormone and human menopausal gonadotrophin has been studied both histologically and immunohistochemically in 30 women undergoing in-vitro fertilization treatment. None had embryo transfer. Patients were randomly allocated to receive luteal phase support with a single dose of human chorionic gonadotrophin. The latter failed to produce any significant enhancement of endometrial structure or secretions. Appropriate glandular morphology was present in a greater proportion of those who were successfully stimulated than those who responded poorly. However, defective secretion of the cycle-dependent component studied, using monoclonal antibody D9B1, was demonstrated in two-thirds of cases regardless of the ovarian response. Early vascular maturation in the stroma was a common finding, and was thus considered as a feature of structural modulation of these endometria.

Introduction Recently, a PET tracer, 9-[ 18 F]fluoropropyl-(+)-dihydrotetrabenazine ([ 18 F]AV-133), targeting vesicular monoamine transporter 2 (VMAT2) in the central nervous system has been reported. It is currently under Phase II clinical trials to establish its usefulness in the diagnosis of neurodegenerative diseases including dementia with Lewy bodies and Parkinson's disease. The radiolabeling of [ 18 F]AV-133, nucleophilic fluorination reaction and potential effects of pseudo-carrier were evaluated by in vivo biodistribution. Methods The preparation of [ 18 F]AV-133 was evaluated under different conditions, specifically by employing different precursors (–OTs or –Br as the leaving group at the 9-propoxy position), reagents (K222/K 2 CO 3 vs. tributylammonium bicarbonate) and solvents (acetonitrile vs. DMSO), reaction temperature and reaction time. With optimized conditions from these experiments, radiosynthesis and purification with solid-phase extraction (SPE) of [ 18 F]AV-133 were performed by an automated nucleophilic [ 18 F]fluorination module. In vivo biodistribution in mice on [ 18 F]AV-133 purified by either HPLC (no-carrier-added) or the SPE method (containing a pseudo-carrier) was performed and the results compared. Results Under a mild fluorination condition (heating at 115°C for 5 min in dimethyl sulfoxide), [ 18 F]AV-133 was obtained in a high yield using either –OTs or –Br as the leaving group. However, the –OTs precursor gave better radiochemical yields (>70%, thin layer chromatography analysis) compared to those of the –Br precursor. The optimized reaction conditions were successfully implemented to an automated nucleophilic fluorination module. Labeling and purification of [ 18 F]AV133 were readily achieved via this automatic module in good radiochemical yield of 21–41% ( n =10) in 40 min. The radiochemical purity was larger than 95%. Biodistribution of SPE-purified product (containing a pseudo-carrier) in mice showed a high striatum/cerebellum ratio (4.18±0.51), which was comparable to that of HPLC-purified [ 18 F]AV-133 (4.51±0.10). Conclusions The formation of [ 18 F]AV-133 was evaluated under different labeling conditions. These improved labeling conditions and SPE purification were successfully implemented into an automated synthesis module. This offers a short preparation time (about 40 min), simplicity in operation and ready applicability for routine clinical operation.

PET of the serotonin transporter (SERT) in the brain is a useful tool for examining normal physiologic functions as well as disease states involving the serotonergic system. The goal of this study was to further develop and refine a series of 4′-fluoroalkoxy–substituted, 18F-radiolabeled SERT imaging agents. 2-(2′-((Dimethylamino)methyl)-4′-(4-18F-fluorobutoxy)phenylthiol)benzenamine (3) and 2-(2′-((dimethylamino)methyl)-4′-(5-18F-fluoropentoxy)phenylthiol)benzenamine (4) were synthesized and evaluated along with 2 previously reported compounds of this series, 2-(2′-((dimethylamino)methyl)-4′-(2-18F-fluoroethoxy)phenylthiol)benzenamine (1) and 2-(2′-((dimethylamino)methyl)-4′-(3-18F-fluoropropoxy)phenylthiol)benzenamine (2). Methods: The in vitro binding affinities of compounds 3 and 4 were determined in monoamine transporter–transfected LLC-PK1 cell homogenates. In vivo localization of the respective 18F-labeled compounds was evaluated by biodistribution studies in male Sprague–Dawley rats. Compound 3 was selected for further examination by autoradiographic and PET studies in rats. Results: The corresponding mesylate precursors of 3 and 4 were radiolabeled with 18F within 75–90 min. Radiochemical yield was 6%−35%, specific activity was 15–170 GBq/μmol, and radiochemical purity was greater than 97% (end of synthesis). The compounds showed subnanomolar binding affinities for SERT (inhibition constants, 0.51 and 0.76 nM, respectively), had brain uptake at 2 min of 1.25 and 0.68 percentage injected dose per gram, respectively, and possessed high target-to-nontarget (hypothalamus-to-cerebellum) ratios at 120 min after injection (6.51 and 5.70, respectively). Autoradiographic studies of 18F-3 showed selective localization in SERT-rich brain regions. PET studies of 18F-3 showed clear localization in the midbrain, thalamus, and striatum. Conclusion: This compound series was found to have potential for producing a suitable 18F-radiolabeled PET radiotracer for SERT. Compound 4, the pentoxy derivative, had the lowest brain uptake and target-to-nontarget ratio. Compound 3, the butoxy derivative, had a lower target-to-nontarget ratio than compounds 1 (ethoxy derivative) and 2 (propoxy derivative). Compounds 1 and 2 both hold promise as SERT radioimaging agents, but because of cost limitations, only compound 2 will be evaluated in further studies.

The cryopreservation of semen used in assisted reproduction procedures was carried out exclusively by a simplified method in which a mixture of semen and cryoprotectant was contained in 1-ml tuberculin syringes and plunged directly into liquid nitrogen. Donor semen samples halved and frozen in syringes and in straws in a controlled-rate freezer showed no significant difference in post-thaw motility (P = 0.217) or survival (P = 0.217) after 30 min. However, after 180 min the survival rate showed a significant reduction in syringes (P = 0.045). A significant difference (P less than 0.00008) in the rate of fertilization of oocytes was seen in IVF cycles using frozen-thawed donor sperm (58/142, 42%) when compared to fresh sperm from husbands (2315/3926, 59%). A significant reduction (P less than 0.00005) in fertilization rate was also observed in the case of supernumerary oocytes in GIFT cycles with the cryopreserved donor sperm (29/132, 22%) compared to the husbands' sperm (239/514, 46%). However, the pregnancy rate following IVF and embryo replacement was the same after fertilization with fresh sperm (75/351, 21%) as opposed to frozen sperm (3/14, 21%). Furthermore, a higher pregnancy rate was observed in GIFT with frozen donor sperm (9/19, 47%) than with fresh sperm from husbands (28/103, 27%), though this was not statistically significant (P = 0.079). These results show this simplified methods of semen cryopreservation to be effective when used in an IVF and GIFT programme, giving pregnancy rates comparable to fresh normospermic semen samples. The method is simple, quick and inexpensive.

Gamete intra-Fallopian transfer (GIFT) was performed in 130 treatment cycles over a 17-month period. In 91% (118/130) of the cycles one or more oocytes were available for insemination in vitro and only GIFT cycles with supernumerary oocytes were included in the present study. Pituitary and ovarian suppression was achieved with buserelin followed by stimulation of multifollicular development by human menopausal gonadotrophin (HMG). Failure of supernumerary oocytes to fertilize was associated with a significantly reduced pregnancy rate (3/23; 13%) compared to cycles where fertilization occurred in vitro (35/95; 37%). These findings demonstrate that the outcome of IVF of supernumerary oocytes may be of particular diagnostic value in couples where the female partner has not conceived following treatment by GIFT after pituitary down-regulation with buserelin and ovarian stimulation with HMG.

Evidence of implantation following either in vitro fertilization and embryo transfer (IVF-ET) or gamete intrafallopian transfer (GIFT) was obtained by collecting blood on days 10, 12, 14, 16, and 18 after oocyte recovery (day 0), and retrospectively measuring human chorionic gonadotropin (hCG). This was done using immunoradiometric assays for hCG, manufactured either by Serono Diagnostics Ltd. (MAIAclone) or Diagnostics Products Corporation (IRMA-count). The analysis of 63 serum samples by both kits showed a good correlation (r = 0.99) but the Serono (y) method gave values which were consistently greater (y = 1.58x + 4.89) than those of the DPC (x) method. A comparison of the hCG profile of singleton pregnancies measured with either the Serono (n = 33) or DPC (n = 22) kits gave a similar relationship. These results suggest that great care must be taken when comparing results from different laboratories using different kits. Also, consideration must be given to the possible loss of a continuous database should a laboratory change kit.

Daily s.c. injections of buserelin were commenced in the mid-luteal phase of the preceding cycle in 118 women undergoing in-vitro fertilization (IVF) and embryo transfer. Ovarian and pituitary suppression was said to have been adequately achieved when serum oestradiol was less than 50 pg/ml, serum LH less than 2.0 IU/l, no ovarian cysts greater than or equal to 10 mm diameter were present and menstruation had occurred. Nine groups of women were retrospectively identified after the administration of buserelin for 12 days according to whether pituitary and ovarian suppression had been achieved or not, and the reason for extended buserelin treatment prior to ovarian stimulation. Upon adequate suppression, patients were grouped in terms of the duration of exposure to buserelin, and ovarian stimulation was then started by daily injections of human menopausal gonadotrophin. There appeared to be no differences in the ovarian response for women down-regulated by day 12, 19 or greater than or equal to 26 days; those women requiring extended buserelin treatment did equally well compared to those women down-regulating quickly, in terms of number of oocytes recovered and fertilization rate. Clinical pregnancy rates per embryo transfer were 27/68(40%), 8/33(26%) and 4/17(24%) for those women down-regulated by days 12, 19 or greater than or equal to 26 respectively, and were not significantly different.

The development of new myocardial perfusion imaging agents for positron emission tomography (PET) may improve the resolution and quantitation of changes in regional myocardial perfusion measurement. It is known that a 68 Ge/ 68 Ga generator can provide a convenient source of PET tracers because of the long physical half-life of 68 Ge (271 days). A new ligand, 7,8-dithia-16,24-diaza-trispiro[5.2.5.2.5.3]pentacosa-15,24-diene, which consists of a N 2 S 2 -chelating core incorporated into three cyclohexyl rings, was prepared. To test feasibility and potential utility, the N 2 S 2 ligand was successfully labeled and tested with 67 Ga (half-life=3.26 day; γ =93.3, 184.6 and 300.2 keV), which showed >92% radiochemical purity. The corresponding "cold" Ga complex was synthesized, and its structure containing a pyramidal N 2 S 2 chloride core was elucidated with X-ray crystallography. In vivo biodistribution of this novel 67 Ga complex, evaluated in normal rats, exhibited excellent heart uptake and retention, with 2.1% and 0.9% initial dose/organ at 2 and 60 min, respectively, after an intravenous injection. Autoradiography was performed in normal rats and in rats that had the left anterior descending coronary artery permanently ligated surgically. Autoradiography showed an even uptake of activity in the normal heart, and there was a distinctively lower uptake in the damaged side of the surgically modified heart. In conclusion, the new N 2 S 2 ligand was readily prepared and labeled with radioactive 67 Ga. Biodistribution in rats revealed high initial heart uptake and relatively high retention reflecting regional myocardial perfusion.

In the UK, the live birth rate after IVF in women aged less than 36 is >25%. The multiple birth rates in these women are excessive (20% to 25%). The perinatal mortality rate is increased significantly with IVF twins and triplets (8/1000 singletons, 20/1000 twins and 34/1000 triplets). Multiple pregnancies and births significantly increase the risks to the mother and the children, adversely affect family life and are economically disadvantageous to the couple and the wider community. The elective transfer of a single fresh embryo, followed if necessary by a single thawed embryo, in women at high risk of a multiple birth does not reduce the live birth rate and all but prevents the conception of twins and triplets.

Background: The breast cancer type 1 susceptibility protein (BRCA1) carries out a primary function in the DNA homologous recombination repair (HRR) pathway and mutations in the BRCA1 gene have been linked to a dramatic incidence in breast and ovarian cancer. BRCA1 associated cancers also lack HRR and are reliant on other DNA repair pathways such as base excision repair (BER) and non-homologous end joining (NHEJ). Three primary proteins involved in BER and NHEJ are Poly(ADP-ribose) Polymerases(PARP)1, 2 and 3, and inhibition of these proteins lead to a synthetic lethality in BRCA1 mutated tumors. Despite unveiling structural and mechanistic properties of BRCA1 and the PARP enzyme super family, PARP inhibition is only effective in relatively low percentages of patients who possess BRCA1 mutations. Therefore, the need for biomarkers to predict patient response to PARP inhibition is highly important. Our lab has developed a radio-iodinated-PARP inhibitor (iodine-125-KX1) capable of quantitative measurements of active PARP enzymes in whole cell assays that can predict the in vitro response to PARP inhibitors. Methods: [125I]KX1 was synthesized through radio-iododestannylation and purified by high-performance liquid chromatography. BRCA1 mutated and non-mutated ovarian and breast cancer cell lines: SNU-251, SKOV3, HCC1937, and MDA-MB-231 were investigated in a live cellular assay with [125I]KX1. Saturation, competitive inhibition, and kinetic assays were performed in all cell lines using [125I]KX1. Western analysis was performed to measure baseline PARP1, Poly(ADP-ribose(PAR), and BRCA1 protein expression. PARP inhibitor efficacy of talazoparib and olaparib was assessed through modified clonogenic assays. [125I]KX1 biodistribution experiments were carried out in mice bearing HCC1937 and MDA-MB-231 tumors to examine PARP in vivo in BRCA1 mutated and non-mutated cancer cell lines. Results: [125I]KX1 was synthesized in high radiochemical purity. Saturation experiments revealed that BRCA1 mutated cancer cell lines had a higher PARP binding potential measured by [125I]KX1. Subtle differences in PARP inhibitor affinity was noticed in the different cell lines through competitive inhibition assays. Kinetic analysis revealed that the SNU-251 had the slowest dissociation kinetics of [125I]KX1. Western analysis confirmed PARP activity measured immunohistochemically by PAR correlated with PARP binding potential measured with [125I]KX1. In vitro PARP inhibitor potency was strongly correlated with PARP binding potential. Biodistribution studies revealed that PARP can be measured quantitatively in vivo. Conclusion: With the utilization of [125I]KX1 we have been able to explore the PARP enzyme family in relation to BRCA1 mutations using in vitro and in vivo breast and ovarian cancer models. Quantitative measurements of PARP enzymes in live cancer cells has not been reported, and provides new insight into understanding the molecular target for PARP inhibitor therapy. Our data shows there is a positive correlation with PARP enzyme binding potential and response to PARP inhibitor therapy in vitro. Differences in PARP binding potential can also be measured in vivo and offer a prognostic biomarker marker for patient's who may receive PARP inhibitor therapy. Citation Format: Mehran Makvandi, Brian P. Lieberman, Kuiying Xu, Redmond-Craig Anderson, Chenbo Zeng, David A. Mankoff, Daniel A. Pryma, Roger A. Greenberg, Robert H. Mach. Predicting response to PARP inhibitors through quantitative measurements of PARP activity in live BRCA1 mutated cells with a radio-iodinated PARP inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C15.

There are conflicting reports as to whether the interval between vasectomy and surgical sperm retrieval (SSR) for intra-cytoplasmic sperm injection (ICSI) is related to clinical pregnancy (CPR), and live birth (LBR), rates. This study aimed to evaluate factors that may influence the outcome of ICSI in males with secondary azoospermia due to previous vasectomy. We analysed the medical records of 198 azoospermic males following vasectomy who underwent percutaneous epididymal sperm aspiration (PESA) and/or testicular sperm extraction (TeSE), between 1997 and 2005 by a single urologist, and whose sperm was subsequently frozen for use in an IVF treatment programme on their partner's behalf. Hundred and forty-four (73%) males had a positive PESA, and the remaining 54 (27%) had a positive TeSE. Forty-four percent of males with no clinical evidence of epididymal distension still had epididymal sperm retrieved successfully. Hundred and twenty-eight patients proceeded with ICSI, and a total of 237 cycles were performed. The CPR and LBR overall were 29 and 27%, respectively. Using logistic regression there was no association between time since vasectomy and CPR (P = 0.17) or LBR (P = 0.31). A history of an attempted reversal of vasectomy did not negatively affect retrieval rates or CPR and LBR. The success of SSR and the outcome of ICSI, using frozen sperm, are independent of male age and time since vasectomy. Epididymal sperm may be retrieved in over 40% of men in whom there is no clinical evidence of epididymal distension.

Objectives Men presenting with primary infertility and azoospermia may be offered surgical sperm retrieval (SSR) as a prelude to intracytoplasmic sperm injection (ICSI). We evaluated sperm retrieval rates in subgroups of men with azoospermia, based on obstructive aetiology, testicular volume and FSH. Methods 106 patients with primary infertility underwent clinical evaluation and SSR with percuataneous epididymal aspiration (PESA) and/or testicular sperm extraction (TeSE) by a single urologist over a five year period. Ten percent of this group (11 patients) had a clear cause of obstruction, congenital absence of the vas deferens (CBAVD), labelled group A. Ninety percent (95 patients) had no definite cause of obstruction, labelled group B. Results All eleven patients in group A had adequate sperm retrieved, compared with 56% of 95 men in group B. Clinical pregnancy and live birth rates were 47% and 44% for group A respectively compared with 21% and 20% for group B. Twenty-one men had testes 10; a significantly lower sperm retrieval rate was seen in this subgroup (29%) compared to men with normal testicular volume and FSH (77%), p =0.0001, which corresponded to a LBR of 28% and 14% respectively. Conclusions In the absence of testicular histology prior to SSR clinical parameters can be used to aid in counselling. Azoospermic males with normal sized testes and normal FSH can expect acceptable numbers of sperm to be retrieved by SSR for ICSI. Less than one third of men with raised FSH and small testes will have successful SSR.

Purpose: Increasing numbers of couples are consulting for infertility and, after some years of investigation and treatment, will become enrolled in in vitro fertilization (IVF) programs. The media have focused on successful outcomes or difficulties in accessing services because of health authority funding guidelines. Couples are often assumed to be highly motivated because of their long prior involvement in treatment. However, little is known about uptake of and continuation through IVF treatment programs.

BACKGROUND: IVF is limited by low success rates and an unacceptably high multiple pregnancy rate. These outcomes would be improved significantly if a single embryo of high viability could be replaced in each treatment cycle, but widespread acceptance of such a policy is hindered by the lack of predictive factors for embryo selection. We have conducted a retrospective clinical study of a novel non-invasive method of embryo selection based on the depletion/appearance of amino acids in the culture medium. METHODS: Fifty-three cycles of IVF treatment using ICSI were studied. Embryos were cultured for 24 h in 4 microl drops of medium containing a physiological mixture of 18 amino acids. The spent medium was analysed for amino acid content by high performance liquid chromatography. RESULTS: The turnover of three amino acids, Asn, Gly and Leu, was significantly correlated with a clinical pregnancy and live birth. These correlations were independent of known predictors, such as female age, basal levels of FSH, embryo cell number and embryo morphological grade. CONCLUSIONS: Non-invasive assay of amino acid turnover has the potential to improve significantly the prospective selection of the most viable embryos, or single embryo, for replacement in an IVF cycle.

Advances in cancer treatment have led to significant improvements in the likelihood of reaching remission and long-term survival for men. Chemo- and radiotherapy-induced infertility are significant treatment side effects. Cryopreservation before the start of treatment enables sperm to be stored, thereby preserving the man's potential fertility. Here, we describe the successful use (with ICSI) of sperm cryopreserved prior to cancer treatment, for a total of 21 years. We believe this to be the longest period of sperm cryopreservation, resulting in a live birth, so far reported in the literature.

Apoptosis during preimplantation development has received much interest because of its potential role in eliminating defective cells. Although development in humans is characterised by a high degree of genetic abnormality, little is known of the regulation of apoptosis in embryos. By PolyA PCR we analysed expression of 11 BCL-2 genes in individual human embryos representative of normal development and in severely fragmented embryos. We demonstrate constitutive expression of BAX in virtually all embryos at all stages of development, and variable expression of BCL2, BCL-XL, BCL-W, MCL-1 BAK, BAD, BOKL, BID, BIK, and BCL-XS. The frequency of expression of pro- and anti-apoptotic BCL-2 members was similar throughout development, except at the two-cell stage where pro-apoptotic genes predominated. Protein expression was confirmed for BCL-2, MCL-1, BCL-X, BAX, BAD, and activated caspase 3. BCL-2 protein was associated with mitochondria but expressed inconsistently in the blastocyst inner cell mass. Consistent differences between morphologically intact and fragmented embryos included the expression of BAK in fragmented but not intact four-cell embryos. Our study addresses the importance of examining single human embryos representative of the viable population for a large number of genes, in order to establish meaningful expression profiles and provide information on overlapping function in a large gene family.

The fertilization and subsequent cryopreservation of donated oocytes have enabled the resulting embryos to be quarantined for a minimum of 6 months, and to be thawed and replaced only after the donor has had a second negative human immunodeficiency virus (HIV) test. In the study described here, a total of 39 women had 56 embryo transfers, and 12 pregnancies (21% per transfer) were achieved. The logic of the protocol for minimizing the risk of infection of the recipient, which is in line with that of semen donation, is presented, together with an argument for the feasibility of such an approach.

Objective To characterize endometrial development in unexplained and tubal factor infertility. Design Prospective study of 20 women with unexplained infertility, 22 with tubal factor infertility, and 21 fertile controls in the midproliferative, periovulatory, and midluteal phases of the menstrual cycle. Setting Reproductive Medicine Department of St. Mary's Hospital, Manchester, United Kingdom. Patient(s) Women awaiting assisted conception. Investigation(s) Serum hormone assays, transvaginal ultrasound, Doppler, and midluteal endometrial biopsies. Main outcome measure(s) Serum levels of E2, P, and LH, endometrial ultrasound morphometry, uterine and subendometrial artery Doppler, and endometrial histology and biochemistry. Result(s) Women with unexplained infertility demonstrated significantly reduced uterine artery flow velocity in all phases, significantly elevated uterine and subendometrial artery impedance in the periovulatory and midluteal phases, and significantly reduced endometrial texture in the midproliferative phase. Women with tubal factor infertility demonstrated significantly reduced uterine artery flow velocity, without a concomitant increase in impedance, and significantly greater expression of endometrial glandular and luminal keratan sulphate. Conclusion(s) Unexplained infertility is associated with a profound impairment of endometrial perfusion that might be amenable to treatment by perfusion enhancers. Tubal factor infertility is associated with endometrial developmental defects that might be corrected by salpingectomy. Endometrial ultrasound and Doppler studies are likely to become a vital tool in the investigation of infertility.

This study analysed the live birth rates in 760 couples referred in 1994 to St Mary's Hospital, Manchester, a non-fee-paying National Health Service (NHS) centre, who had waited for up to 4 years for IVF treatment. These live birth rates were compared with those of 199 couples referred at a similar time to Manchester Fertility Services, a fee-paying unit, where they received IVF treatment shortly after referral. The waiting time was advantageous in that 17.8% (135 of 760) of the couples referred to St Mary's Hospital conceived without IVF treatment, 60% within one year of referral. However, the waiting time was detrimental to women aged 30–34 in whom treatment was delayed by 3–4 years. Only 26.8% (204 of 760) of couples originally referred eventually received NHS-funded IVF treatment at St Mary's. A waiting time not exceeding 18 months would allow most spontaneous conceptions and reduce the adverse effect of prolonged waiting on the take-up rate for treatment and on the chance of success in the older women.

A total of 67 semen donors (62 Caucasian and five non-Caucasian) were tested for the presence of immunoglobulin G antibodies to cytomegalovirus (CMV). Ten (16%) of the Caucasian donors tested positive for CMV, while four (80%) of the non-Caucasian donors were positive. A total of 131 women receiving donor insemination (114 Caucasian and 17 non-Caucasian) were tested. Of these, 43 (38%) of the Caucasian recipients tested positive for CMV, while 16 (94%) of the non-Caucasian recipients were positive. There was a significant association between the incidence of positive tests and the age of the Caucasian recipients.

BACKGROUND: Ovarian failure is a common sequel to chemo/radiotherapy in patients successfully treated for cancer. Harvesting, cryopreserving and subsequently re-implanting ovarian cortical grafts can be used to reestablish reproductive potential in women with cancer. The safety issue, however, is of great concern because residual disease in autografted ovarian tissues might cause recrudescence of disease. METHODS: A total of 30 non-obese diabetic severe combined immunodeficient (NOD/LtSz-SCID) mice were individually xenografted s.c. with frozen–thawed ovarian tissue from 18 patients with lymphoma [13 Hodgkin’s lymphoma (HL) and 5 nonHodgkin’s lymphoma (NHL)]. The animals were autopsied at 16 weeks, or earlier if cachectic. The xenograft, liver, spleen, sternum, para-aortic lymph nodes and thymus were prepared for histology, immunohistochemistry and human DNA microsatellite analysis. RESULTS: None of the animals grafted with ovarian tissue from lymphoma patients developed disease. However, all 3 animals grafted with lymph node tissue from an NHL patient developed B-cell lymphomas that were confirmed as human in origin by DNA microsatellite analysis. CONCLUSION: Ovarian tissue harvested before high-dose chemotherapy for HL or NHL may not carry a risk of disease transmission by autotransplantation, although the possibility is difficult to exclude completely.

Background There have been reports suggesting that children born after in-vitro fertilisation by intracytoplasmic sperm injection (ICSI) are at increased risk of neurodevelopmental delay. We have undertaken a case-control study of this issue.Methods We studied 208 singleton children conceived by ICSI and a control group of 221 normally conceived singleton children. Children were recruited from 22 fertility centres and local nurseries throughout the UK. Controls were selected to match cases as closely as possible for social class, maternal educational attainment, region, sex, and race. The primary outcome measure was neurodevelopmental scoring; secondary measures were perinatal outcomes, postnatal health, and congenital abnormalities. A single examiner assessed all the children.Findings A follow-up rate of 90% for the ICSI group was achieved at a mean age of 17 months. No difference between the study children and controls was found in mean neurodevelopmental scores (98.08 [SD 10.93] vs 98.69 [9.99]) or any subscales on the Griffiths' scales of mental development. Perinatal outcome was similar apart from a higher rate of caesarean section (73 [35.1%] vs 53 [24.0%], p=0.015) and a lower mean birthweight (3163 [SD 642] vs 3341 [606] g, p=0.013) in the study group. Rates of major congenital abnormality were also similar overall (ten [4.8%] study vs ten [4.5%] control), although there were significantly more congenital anomalies among children born to fathers with oligozoospermia than in other children.Interpretation This population study did not show any significant difference between children conceived after ICSI and their naturally conceived peers in terms of physical health and development.