A brief history of technical developments in intracytoplasmic sperm injection (ICSI). Dedicated to the memory of J.M. Cummins.

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technology for treatment of severe male infertility introduced into clinical practice in 1992. This review provides a brief history of the development of ICSI by acknowledging major developments in the field. The review addresses key developments in pre-clinical and early studies, how ICSI compares with in vitro fertilisation, long-term consequences, how the mechanistic approach to ICSI has changed in both manual and semi-automated approaches, and how sperm selection procedures are integrated into ICSI. From the beginnings using animal models in the 1960-1970s, the development of ICSI is a remarkable and transformative success story. Indeed, its broad use (70% of cycles globally) exceeds the need required for treating infertile males, and this remains a controversial issue. There remain questions around the long-term health impacts of ICSI. Furthermore, advances in automation of the ICSI procedure are occurring. An estimated 6million children have been born from the ICSI procedure. With further automation of sperm selection technologies, coupled with automation of the injection procedure, it is likely that the proportion of children born from ICSI will further increase.

Oocyte and embryo evaluation by AI and multi-spectral auto-fluorescence imaging: Livestock embryology needs to catch-up to clinical practice.

A highly accurate 'non-invasive quantitative embryo assessment for pregnancy' (NQEAP) technique that determines embryo quality has been an elusive goal. If developed, NQEAP would transform the selection of embryos from both Multiple Ovulation and Embryo Transfer (MOET), and even more so, in vitro produced (IVP) embryos for livestock breeding. The area where this concept is already having impact is in the field of clinical embryology, where great strides have been taken in the application of morphokinetics and artificial intelligence (AI); while both are already in practice, rigorous and robust evidence of efficacy is still required. Even the translation of advances in the qualitative scoring of human IVF embryos have yet to be translated to the livestock IVP industry, which remains dependent on the MOET-standardised 3-point scoring system. Furthermore, there are new ways to interrogate the biochemistry of individual embryonic cells by using new, light-based methodologies, such as FLIM and hyperspectral microscopy. Combinations of these technologies, in particular combining new imaging systems with AI, will lead to very accurate NQEAP predictive tools, improving embryo selection and recipient pregnancy success.

The contribution of mitochondrial respiratory complexes to the production of reactive oxygen species.

This work was focused on distinguishing the contribution of mitochondrial redox complexes to the production of reactive oxygen species (ROS) during cellular respiration. We were able to accurately measure, for the first time, the basal production of ROS under uncoupled conditions by using a very sensitive method, based on the fluorescent probe dichlorodihydrofluorescein diacetate. The method also enabled the detection of the ROS generated by the oxidation of the endogenous substrates in the mitochondrial preparations and could be applied to both mitochondria and live cells. Contrary to the commonly accepted view that complex III (ubiquinol:cytochrome c reductase) is the major contributor to mitochondrial ROS production, we found that complex I (NADH-ubiquinone reductase) and complex II (succinate-ubiquinone reductase) are the predominant generators of ROS during prolonged respiration under uncoupled conditions. Complex II, in particular, appears to contribute to the basal production of ROS in cells.

Bcl-2 and mitochondrial oxygen radicals. New approaches with reactive oxygen species-sensitive probes.

Investigations into the capacity of the Bcl-2 protein to prevent apoptosis have targeted mitochondria as key sites of the preventative action accorded by Bcl-2 to cells. Using novel approaches with fluorescence probes and autofluorescence detection of endogenous NAD(P)H, we have examined the effects of expressing Bcl-2 in the Bcl-2 negative Burkitt's lymphoma cell line Daudi. We evaluated for the first time the effect of Bcl-2 expression on the intracellular distribution and production of hydrogen peroxide, under basal conditions and after treatment with apoptosis inducing agents, ceramide analogs and tumor necrosis factor (TNF)-alpha. Increased availability of mitochondrial NAD(P)H was detected in Bcl-2-expressing cells and was correlated with an increased constitutive mitochondrial production of hydrogen peroxide. Although production of hydrogen peroxide was increased by either C(6)-ceramide or TNF-alpha in Bcl-2 negative Daudi cells commensurate with the early phases of apoptosis, this increase did not occur in Bcl-2-expressing cells. Thus, Bcl-2 appears to allow cells to adapt to an increased state of oxidative stress, fortifying the cellular anti-oxidant defenses and counteracting the radical overproduction imposed by different cell death stimuli. Furthermore, we report altered cytological features of mitochondria during the early phases of apoptosis induced by C(6)-ceramide and TNF-alpha. In particular, mitochondria changed in appearance, clustering in the perinuclear region and Bcl-2 expression prevented these changes from occurring.

Mitochondria and cells produce reactive oxygen species in virtual anaerobiosis: relevance to ceramide-induced apoptosis.

Observations of apoptosis in virtual anaerobiosis have raised doubts on the significance of reactive oxygen species in the cascade of events of programmed cell death. This work presents evidence that cells and mitochondrial preparations produce similar levels of hydrogen peroxide under either aerobic or virtually anaerobic conditions. These levels are relevant to the increased production of radicals induced by a ceramide analog that promotes apoptosis. This ceramide acts at center o of mitochondrial complex III.

The interaction of Q analogs, particularly hydroxydecyl benzoquinone (idebenone), with the respiratory complexes of heart mitochondria.

We have studied the interaction of idebenone (2,3-dimethoxy-5-methy-6-(10-hydroxy)decyl-1,4-benzoquinone) with the energy-conserving complexes of the respiratory chain in beef heart mitochondria and compared its energetic efficiency with that of other analogs of coenzyme Q. Idebenone is a very effective substrate for succinate:Q reductase and ubiquinol:cytochrome c reductase, but it is clearly a poor substrate for NADH:Q reductase (complex I). Indeed, idebenone is a strong inhibitor of both the redox and proton pumping activity of complex I, showing effects in part similar to those of coenzyme Q-2. However, the mechanism of idebenone interaction with complex I may be different from that of Q-2 because of its different sensitivity to inhibitors. The possible relevance of the present findings to the therapeutic use of idebenone is discussed.

Quantitative analysis of two pyridinium metabolites of haloperidol in patients with schizophrenia.

OBJECTIVE: A pyridinium metabolite (HPP+) of the neuroleptic drug haloperidol has been identified in rats and in the urine of patients. The purpose of this study was to measure the steady-state blood and plasma concentrations and daily urinary excretion of HPP+ in patients treated with haloperidol. METHODS: HPP+ was measured by HPLC with fluorescence detection. The chromatograms also revealed the presence of a previously unknown pyridinium species, which was identified in urine by liquid chromatography/mass spectrometry/mass spectrometry as 4-(4-chlorophenyl)-1-4-(4-fluorophenyl)-4-hydroxybutylpyridinium (RHPP+). Concentrations of RHPP+ were then measured by HPLC. RESULTS: The steady-state concentrations of HPP+ or RHPP+ in blood and plasma from 34 patients were virtually identical. The plasma concentrations of each metabolite were related to the daily dose of haloperidol and to its plasma concentrations. Nonlinearity in the elimination of RHPP+ was suggested by the increase in the ratio between RHPP+ and HPP+ plasma concentrations with dose or steady-state concentrations of haloperidol. The concentrations of RHPP+ in plasma and urine generally exceeded those of HPP+; the ratio between them in plasma ranged from 0.9 to 14.1. The daily urinary excretion of HPP+ and RHPP+ accounted for 0.40% +/- 0.18% and 2.3% +/- 1.4% of the haloperidol dose, respectively. The renal clearance of each species was 4.5 +/- 2.5 and 11.3 +/- 5.3 L/hr, respectively. CONCLUSIONS: The presence of these pyridinium species in humans raises the concern that they may be neurotoxic in a manner similar to the dopaminergic pro-neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

The actions of a conformationally restricted analogue of aspartic acid on mammalian spinal neurones.

The effects of the 4 stereoisomers of 1-aminocyclopentane-1,2-dicarboxylic acid (CPA), a conformationally restricted analogue of aspartic acid, have been examined on spinal dorsal horn neurones of the rat in vivo. Unlike the corresponding 1,3-dicarboxylate compounds which are glutamate analogues and markedly excitatory, the CPA isomers had no evident excitatory actions of their own. Three were weak and non-specific antagonists of other amino acid-induced excitations, while the (+)-trans isomer had a slight potentiating effect.

Bursting response to current-evoked depolarization in rat CA1 pyramidal neurons is correlated with lucifer yellow dye coupling but not with the presence of calbindin-D28k.

Calbindin-D28k (CaBP) immunohistochemistry has been combined with electrophysiological recording and Lucifer Yellow (LY) cell identification in the CA1 region of the rat hippocampal formation. CaBP is shown to be contained within a distinct sub-population of CA1 pyramidal cells which is equivalent to the superficial layer described by Lorente de Nó (1934). The neurogenesis of these CaBP-positive neurons occurs 1-2 days later than the CaBP-negative neurons in the deep pyramidal cell layer, as shown by 3H-thymidine autoradiography. No correlation could be found between the presence or absence of CaBP and the type of electrophysiological response to current-evoked depolarizing pulses. The latter could be separated into bursting or non-bursting types, and the bursting-type response was nearly always found to be associated with the presence of LY dye coupling. Furthermore, when dye coupling involved three neurons, a characteristic pattern was observed which may represent the coupling of phenotypically identical neurons into distinct functional units within the CA1 pyramidal cell layer. In this particular case the three neurons were all likely to be CaBP-positive.

Electrophysiological properties of rat CA1 pyramidal neurones in vitro modified by changes in extracellular bicarbonate.

1. Intracellular recordings were made from the somata of CA1b hippocampal pyramidal neurones in vitro and the concentration of bicarbonate ion ([HCO3-]o) in the artificial cerebrospinal fluid (ACSF) was varied by substitution for Cl-. 2. Reducing [HCO3-]o from 26 mM (standard ACSF) to 8.6 mM or raising it to 72 mM had only minor effects on resting membrane potential but input resistance was reduced and increased, respectively. Threshold for Na(+)-dependent action potential generation was raised during the perfusion with low-HCO3- ACSF and lowered during perfusion with high-HCO3- ACSF. In tetrodotoxin-poisoned neurones where presumed Ca2(+)-dependent potentials could be elicited in standard ACSF, perfusion with high-HCO3- ACSF lowered the activation threshold. Where no Ca2(+)-dependent spikes could be elicited in standard ACSF, perfusion with high-HCO3- ACSF caused their appearance. Ca2(+)-dependent spikes could not be evoked during perfusion with low-HCO3- ACSF. 3. Depolarizing current pulses evoked two basic patterns of action potential discharge under standard [HCO3-]o conditions. Conversion between the two types was possible by varying [HCO3-]o. In those neurones which fired a train of fast Na(+)-dependent spikes in standard ACSF, perfusion with high-HCO3- ACSF usually led to the development of burst discharges. A smaller number of neurones responded to depolarizing current with an initial burst of three to six action potentials; the bursts were attenuated in low-HCO3- ACSF and replaced by a repetitive spike discharge. Frequency accommodation of spike discharge in response to depolarizing current pulses and the after-hyperpolarization following a current-evoked discharge were usually both attenuated in low-HCO3- ACSF and enhanced in high-HCO3- ACSF. 4. Orthodromically evoked excitatory postsynaptic potentials (EPSPs) and early and late inhibitory postsynaptic potentials (IPSPs) were reduced in amplitude during perfusion with low-HCO3- ACSF. In high-HCO3- ACSF, EPSP amplitude and duration increased, the latter reflecting a positive shift in the reversal potential of the early IPSP consequent upon reduced [Cl-] in high-HCO3- ACSF. The late IPSP was, however, unaffected. 5. Responses to ionophoretically applied excitatory amino acids were enhanced in high-HCO3- ACSF and depressed in low-HCO3- ACSF. 6. Perfusion with high-HCO3- ACSF was associated with the development of epileptiform activity. Spontaneous or synaptically evoked bursts of action potentials were indistinguishable and could be blocked by N-methyl-D-aspartate antagonists.(ABSTRACT TRUNCATED AT 400 WORDS)

Umbilical cord knots and encirclements.

Although cord knots and/or encirclements account for 1 in 10 stillbirths of infants weighing 2,500 g or more, no problem due to this cause was encountered in a prospective study of 1,115 vaginal deliveries. In this study there were 6 cases of cord knot (0.5%) and 158 of cord encirclement (14.2%). The range of cord length was 27-122 cm, the 10th, 50th and 90th percentiles being 40, 52 and 69 cm respectively. In this study there was no clinical warning (fetal distress) of cord encirclement or knot during pregnancy, labour or delivery.

Synthesis, resolution, and absolute configuration of the isomers of the neuronal excitant 1-amino-1,3-cyclopentanedicarboxylic acid.

The endogenous amino acids glutamate and aspartate depolarize mammalian neurons to produce excitation, and the rigid glutamate analogue 1-amino-1,3-cyclopentanedicarboxylic acid also has this effect. This compound exists as two pairs of geometric isomers, and in the present study the absolute configuration of the four isomers is assigned. The known (+)-S and (-)-R isomers of 3-oxocyclopentanecarboxylic acid were used as the basis for the synthesis. The cis and trans amino acids were obtained by fractional crystallization. Spectral data, including optical rotation, circular dichroism, and 13C nuclear magnetic resonance, are presented. The compounds were evaluated as excitants by microiontophoretic ejection into the dendritic region of impaled CA1 pyramidal neurons of rat hippocampal slices. One isomer, cis-1R,3R, mimicked completely the actions elicited by N-methyl-D-aspartic acid; the other three isomers were alpha-kainic acid like.

Structural requirements for activation of excitatory amino acid receptors in the rat spinal cord in vitro.

The conformational requirements for activation of N-methyl-D-aspartate (NMDA) and quisqualate (QUIS) excitatory amino acid receptors on rat spinal neurones in vitro have been examined using a number of conformationally restricted compounds related to L-glutamate (L-GLU). The excitants were assigned to a receptor type on the basis of their susceptibility to blockade by D (-)-2-amino-5-phosphonvalerate (DAPV) and kynurenate (KYNA). When iontophoretically applied to unidentified neurones in the dorsal horn of spinal cord slices maintained in vitro, three of the isomers of 1-amino-1,3-cyclopentane dicarboxylate (ACPD) evoked excitations which were DAPV-sensitive and therefore were probably elicited via NMDA receptors. The fourth isomer (D-trans-(1R,3S)-ACPD) resembled quinolinate (QUIN) in its actions, and differed from both NMDA and QUIS. Several pyridine derivatives in addition to QUIN were tested, and both the 2,5- and 2,6-pyridine dicarboxylates evoked excitations which, like those produced by QUIS and L-GLU, were largely unaffected by both DAPV and KYNA and thus appeared due to activation of the QUIS receptor. 2,4-Pyridine dicarboxylate acted as a weak and unselective antagonist of amino acid-induced excitations. The results support an earlier conclusion that compounds reacting with the NMDA receptor do so in an extended configuration whereas the QUIS receptor has a more folded template. The possibility that QUIN reacts with a receptor different from those activated by other amino acids is considered.

The actions of cyclopentane analogues of glutamic acid at binding sites for kainic and glutamic acids.

The actions of the four isomers of 1-amino-1, 3-cyclopentane dicarboxylate (ACPD), a conformationally restricted analogue of glutamate, have been examined for their ability to displace radiolabelled kainate and glutamate from their binding sites on membranes prepared from rat brain. All four of the isomers reduced specific kainate binding, and all enhanced that of glutamate although one (D-(-)-cis-(1R,3R)-ACPD) was significantly more active in this respect than were the other three. The results are discussed in terms of the pharmacological effects of the isomers of ACPD on single neurones and the possible structural requirements of the NMDA and kainate receptors.

Survey of hand symptoms in pregnancy.

In a study of 1216 pregnancies, 427 (35%) patients reported hand symptoms. Symptoms of the same quality and distribution were reported by 40 (30%) of 132 control subjects within the previous year, and although invariably mild, these symptoms suggest that pregnancy may aggravate a pre-existing condition. Fewer than 20% of the 427 affected patients described a classic median-nerve symptom distribution (carpal tunnel syndrome), while 12% of patients described an ulnar-nerve distribution, which is thought to represent a genuine and previously underestimated occurrence of ulnar-nerve neuropathy in pregnancy. In 69% of patients, hand symptoms were generalized. Most symptoms were bilateral, commenced in the third trimester and resolved soon after delivery. There was a significant correlation of hand symptoms in pregnancy with the presence of preeclampsia, tight rings, the weight at confinement, the birth weight and a history of premenstrual bloating. Operative intervention was not required for any patient.

The action of quinolinate in the rat spinal cord in vitro.

The responses of dorsal horn neurones to the excitatory amino acids quisqualate, kainate, N-methyl-D-aspartate (NMDA), and quinolinate have been examined in an in vitro preparation of the rat spinal cord. The antagonism of these responses by iontophoretically applied D-(-)-2-amino-5-phosphonovalerate (DAPV), kynurenate, and acridinate was tested, and the results were compared with data obtained from the spinal cord in vivo. The pattern of antagonism was similar in both preparations, although the potencies of agonists and antagonists were found to be significantly greater in vitro. The antagonism of amino acid induced firing of neurones was also recorded during the application of DAPV and kynurenate in the bathing medium. Dose-response curves and IC50 values were determined for these antagonists against all four agonists. The responses to quinolinate were antagonized differently from those to NMDA, quisqualate, or kainate, suggesting that quinolinate does not act specifically through the NMDA receptor as it does in other regions, nor does it appear to act via two or more of the three archetypal amino acid receptors. These findings suggest that a fourth amino acid receptor responsible for quinolinate's action in the spinal cord may exist.

Excitation of rat hippocampal neurones by the stereoisomers of cis- and trans-1-amino-1,3-cyclopentane dicarboxylate.

Intracellular recordings were obtained from rat hippocampal neurons during the microiontophoretic ejection of the stereoisomers of cis- and trans-1-amino-1,3-cyclopentane dicarboxylate into the dendritic region (stratum radiatum) of the impaled cells. L-(+)-cis-1-Amino-1,3-cyclopentane dicarboxylate, D(+)-trans-1-amino-1,3-cyclopentane dicarboxylate, and L-(-)-trans-1-amino-1,3-cyclopentane dicarboxylate all evoked patterns of excitation resembling that elicited by kainate. All of these responses were unaffected by D-(-)-2-amino-5-phosphonovalerate but were antagonized at comparable currents by kynurenate. The excitation produced by D-(-)-cis-1-amino-1,3-cyclopentane dicarboxylate was similar to that evoked by N-methyl-D-aspartate. At low ejection currents a slow depolarization triggered rhythmic burst firing, each burst consisting of a depolarizing shift in membrane potential upon which were superimposed four to five action potentials. These responses were antagonized both by D-(-)-2-amino-5-phosphonovalerate and by kynurenate. The results are discussed with respect to the conformational requirements considered to be necessary for interaction at the kainate and N-methyl-D-aspartate receptors on CA1 pyramidal neurones. It is important to note that the isopropylene side chain of kainate is absent from the 1-amino-1-3-cyclopentane dicarboxylate molecule.

The N-methyl-D-aspartate receptor and burst firing of CA1 hippocampal pyramidal neurons.

Previous intracellular investigations in the rat hippocampus have demonstrated that N-methyl-D-aspartate, ibotenate and 2,3-pyridine dicarboxylate (quinolinate) all evoke burst firing of CA1 pyramidal neurons, whereas kainate and quisqualate, which are thought to react with different receptors, do not. The purpose of the present study has been to investigate the ability of a series of compounds either to trigger burst firing or to antagonize this pattern of excitation. We report here that N-methyl-L-aspartate, 1,2-benzene dicarboxylate (phthalate) and methylene succinate (itaconate) are also capable of evoking burst firing. The results of this investigation suggest that since both quinolinate and phthalate are rigid planar molecules and only the 2 and 3 positioning of the carboxylates of pyridine was active, a cis configuration of the carboxyls with respect to the 2,3 carbon bond appears to be necessary for excitation. While a nitrogen atom is not necessary for activity (this is absent in phthalate and itaconate) a third functional group, bearing at least a partial positive charge, and in a position alpha to one of the carboxyl groups is required. The requirements for pyridine derivatives to trigger burst firing is similar to that reported as necessary for evoking convulsions and neurotoxicity after intrahippocampal infusion and a correlation between N-methyl-D-aspartate-like burst firing and depolarization and this neuropathology is considered. An important observation has been that the addition of a benzene ring to either quinolinate or phthalate to yield 2,3-quinoline dicarboxylate and 2,3-napthalene dicarboxylate, respectively, converted these excitants into antagonists of burst firing.(ABSTRACT TRUNCATED AT 250 WORDS)