Shimon Vega in the eyes of his students and postdocs.

Professor Shimon Vega (1943-2021) of the Weizmann Institute of Science passed away on the 16-th of November. Shimon Vega established theoretical frameworks to develop and explain solid-state nuclear magnetic resonance (NMR) and dynamic nuclear polarization (DNP) techniques and methodologies. His departure left a profound mark on his many students, postdocs, and colleagues. Shortly after his passing, we all assembled spontaneously for an international online meeting to share our reflections and memories of our experiences in Shimon's lab and how they affected us deeply during that period of timeand throughout our scientific careers. These thoughts and feelings were put here into writing.

Low-Field Nuclear Polarization Using Nitrogen Vacancy Centers in Diamonds.

It was recently demonstrated that bulk nuclear polarization can be obtained using nitrogen vacancy (NV) color centers in diamonds, even at ambient conditions. This is based on the optical polarization of the NV electron spin, and using several polarization transfer methods. One such method is the nuclear orientation via electron spin locking (NOVEL) sequence, where a spin-locked sequence is applied on the NV spin, with a microwave power equal to the nuclear precession frequency. This was performed at relatively high fields, to allow for both polarization transfer and noise decoupling. As a result, this scheme requires accurate magnetic field alignment in order preserve the NV properties. Such a requirement may be undesired or impractical in many practical scenarios. Here we present a new sequence, termed the refocused NOVEL, which can be used for polarization transfer (and detection) even at low fields. Numerical simulations are performed, taking into account both the spin Hamiltonian and spin decoherence, and we show that, under realistic parameters, it can outperform the NOVEL sequence.

Heteronuclear DNP of protons and deuterons with TEMPOL.

Dynamic nuclear polarization (DNP) experiments on samples with several types of magnetic nuclei sometimes exhibit "cross-talk" between the nuclei, such as different nuclei having DNP spectra with similar shapes and enhancements. In this work we demonstrate that while at 20 K the DNP spectra of (1)H and (2)H nuclei, in a sample composed of 50% v/v (1)H2O/DMSO-d6 and containing 40 mM TEMPOL, are different and can be analyzed using the indirect cross effect (iCE) model, at 6 K the DNP spectra of both (1)H and (2)H nuclei become identical. In addition we experimentally demonstrate that there exists an efficient polarization exchange between the two nuclear pools at this temperature. Both of these results are hallmark predictions of the thermal mixing (TM) formalism. However, the origin of these observations cannot, in our case, be explained using the standard TM formalism, as in our sample the electron reservoir cannot be described by a single non-Zeeman spin temperature, which is a prerequisite of TM. This conclusion follows from the analysis of the electron electron double resonance (ELDOR) experiments on our sample and is similar to the previously published results. Consequently, another mechanism must be used in order to explain these "cross-talk" effects. The heteronuclear cross effect (hnCE) DNP mechanism, previously introduced based on the simulations of the spin evolution in small model systems, results in "cross-talk" effects between two types of nuclei that are similar to the experimental ones seen in this work. In particular we show that the hnCE mechanism exhibits polarization transfer between the nuclei and that there exists a clear relationship between the steady state polarizations of the two types of nuclei which may, in the future, be correlated with the phenomenon observed in the two types of bulk nuclear signals in samples during DNP experiments. It is suggested that the hnCE electrons are a possible source for the process that equalizes the bulk enhancements of the (1)H and (2)H nuclei and are responsible for the observed cross-talk effects.

Effects of the electron polarization on dynamic nuclear polarization in solids.

Dynamic Nuclear Polarization (DNP) experiments on solid dielectrics can be described in terms of the Solid Effect (SE) and Cross Effect (CE) mechanisms. These mechanisms are best understood by following the spin dynamics in electron-nuclear and electron-electron-nuclear model systems, respectively. Recently it was shown that the frequency swept DNP enhancement profiles can be reconstructed by combining basic SE and CE DNP spectra. However, this analysis did not take into account the role of the electron spectral diffusion (eSD), which can result in a dramatic loss of electron polarization along the EPR line. In this paper we extend the analysis of DNP spectra by including the influence of the eSD process on the enhancement profiles. We show for an electron-electron-nuclear model system that the change in nuclear polarization can be caused by direct MW irradiation on the CE electron transitions, resulting in a direct CE (dCE) enhancement, or by the influence of the eSD process on the spin system, resulting in nuclear enhancements via a process we term the indirect CE (iCE). We next derive the dependence of the basic SE, dCE, and iCE DNP spectra on the electron polarization distribution along the EPR line and on the MW irradiation frequency. The electron polarization can be obtained from ELDOR experiments, using a recent model which describes its temporal evolution in real samples. Finally, DNP and ELDOR spectra, recorded for a 40 mM TEMPOL sample at 10-40 K, are analyzed. It is shown that the iCE is the major mechanism responsible for the bulk nuclear enhancement at all temperatures.

The electron depolarization during dynamic nuclear polarization: measurements and simulations.

Dynamic nuclear polarization is typically explained either using microscopic systems, such as in the solid effect and cross effect mechanisms, or using the macroscopic formalism of spin temperature which assumes that the state of the electrons can be described using temperature coefficients, giving rise to the thermal mixing mechanism. The distinction between these mechanisms is typically made by measuring the DNP spectrum - i.e. the nuclear enhancement profile as a function of irradiation frequency. In particular, we have previously used the solid effect and cross effect mechanisms to explain temperature dependent DNP spectra. Our past analysis has however neglected the effect of depolarization of the electrons resulting from the microwave (MW) irradiation. In this work we concentrate on this electron depolarization process and perform electron-electron double resonance (ELDOR) experiments on TEMPOL and trityl frozen solutions, using a 3.34 Tesla magnet and at 2.7-30 K, in order to measure the state of the electron polarization during DNP. The experiments indicate that a significant part of the EPR line is affected by the irradiation due to spectral diffusion. Using a theoretical framework based on rate equations for the polarizations of the different electron spin packets and for those of the nuclei we simulated the various ELDOR line-shapes and reproduced the MW frequency and irradiation time dependence. The obtained electron polarization distribution cannot be described using temperature coefficients as required by the classical thermal mixing mechanism, and therefore the DNP mechanism cannot be described by thermal mixing. Instead, the theoretical framework presented here for the analysis of the ELDOR data forms a basis for future interpretation of DNP spectra in combination with EPR measurements.

Direct enzyme-substrate affinity determination by real-time hyperpolarized (13)C-MRS.

A specialized kinetic analysis of real-time hyperpolarized [1,1,2,2-D4, 1-(13)C]choline (13)C-magnetic resonance spectroscopy enabled the determination of initial rates of metabolic enzyme activity (choline oxidase), enzyme-substrate affinity (Km), and inhibition. In a clinical MRI scanner, metabolite levels lower than 16 µM were detected at a temporal resolution of 1 s.

Dynamic nuclear polarization using frequency modulation at 3.34 T.

During dynamic nuclear polarization (DNP) experiments polarization is transferred from unpaired electrons to their neighboring nuclear spins, resulting in dramatic enhancement of the NMR signals. While in most cases this is achieved by continuous wave (cw) irradiation applied to samples in fixed external magnetic fields, here we show that DNP enhancement of static samples can improve by modulating the microwave (MW) frequency at a constant field of 3.34 T. The efficiency of triangular shaped modulation is explored by monitoring the (1)H signal enhancement in frozen solutions containing different TEMPOL radical concentrations at different temperatures. The optimal modulation parameters are examined experimentally and under the most favorable conditions a threefold enhancement is obtained with respect to constant frequency DNP in samples with low radical concentrations. The results are interpreted using numerical simulations on small spin systems. In particular, it is shown experimentally and explained theoretically that: (i) The optimal modulation frequency is higher than the electron spin-lattice relaxation rate. (ii) The optimal modulation amplitude must be smaller than the nuclear Larmor frequency and the EPR line-width, as expected. (iii) The MW frequencies corresponding to the enhancement maxima and minima are shifted away from one another when using frequency modulation, relative to the constant frequency experiments.

The use of ICSI with fresh and cryopreserved electroejaculates from psychogenic anejaculatory men.

BACKGROUND: Electroejaculation has become an accepted form of semen procurement in men suffering from anejaculation. However, sperm in these ejaculates often exhibit low motility. In such cases, ICSI is offered to improve the possibility of successful pregnancy. Here we evaluate the fertilizing potential, using ICSI, of fresh and cryopreserved sperm obtained by transrectal electroejaculation from patients with psychogenic anejaculation. METHODS: A total of 25 men suffering from psychogenic anejaculation underwent 37 sessions of electroejaculation in combination with ICSI. In 17 patients fresh sperm (29 cycles, group I) was used, and in the other eight patients cryopreserved sperm (10 cycles, group II) was used. RESULTS: A total of 155 oocytes were injected with fresh sperm with a fertilization rate of 55% (85/155). The pregnancy rate was 10% (3/29) per cycle. A total of 94 oocytes were injected with frozen-thawed sperm with a fertilization rate of 50% (47/94). The pregnancy rate was 40% (4/10) per cycle. CONCLUSIONS: The fertilization and pregnancy rates with cryopreserved sperm from electroejaculation are at least as good as those of freshly obtained sperm. Therefore, when motile sperm is found in the thawed ejaculate, additional electroejaculation can be avoided.

The development of an oocyte-containing follicle during gonadotrophin-releasing hormone agonist administration.

Administration of gonadotrophin-releasing hormone (GnRH) agonist in a 29 year old woman with infertility due to ovulatory dysfunction resulted in the development of several ovarian cysts. After human chorionic gonadotrophin (HCG) was injected, the cysts were aspirated and one mature oocyte was retrieved. Intracytoplasmic sperm injection (ICSI) was performed and the resulting embryo was transferred. A singleton pregnancy was obtained and a healthy baby was born at 36 weeks of gestation. Because GnRH agonist-derived cysts may contain oocytes, we suggest that when the growth of cysts is accompanied by high concentrations of oestradiol, the administration of HCG may be useful to achieve oocyte maturation and advance IVF treatment.

Electroejaculation before chemotherapy in adolescents and young men with cancer.

OBJECTIVE: To evaluate the outcome of repeated electroejaculation for obtaining semen from adolescents and young men before initiation of anticancer therapies. DESIGN: Retrospective clinical study. SETTING: Bikur Cholim Hospital, Jerusalem, Israel. PATIENT(S): Six young male patients (average age, 18+/-3 years) with diagnosed cancer who underwent 12 procedures of electroejaculation before chemotherapy. INTERVENTION(S): Transrectal electroejaculation. Semen was cryopreserved in small aliquots. MAIN OUTCOME MEASURE(S): Semen analysis. RESULT(S): In all patients, semen was obtained by electroejaculation. Sperm count and motility were relatively low; mean values were 16 x 10(6) (range, 0--45 x 10(6)) and 14% (range, 0--53%) respectively. CONCLUSION(S): If necessary, electroejaculation can be performed in adolescents, and sperm may be obtained by repeated treatments over a short period.

Nocturnal sperm emission in men with psychogenic anejaculation.

OBJECTIVE: To evaluate sperm characteristics and fertilization potential in sperm obtained from nocturnal emission in men with psychogenic anejaculation. DESIGN: Retrospective study. SETTING: In Vitro Fertilization Unit, Bikur Cholim Hospital, Jerusalem, Israel. PATIENT(S): Six men with psychogenic anejaculation. INTERVENTION(S): Nocturnal emission, electroejaculation, sperm cryopreservation, and assisted reproduction technologies. MAIN OUTCOME MEASURE(S): Semen analysis, intracytoplasmic sperm injection (ICSI), fertilization rates. RESULT(S): In four patients, the concentration and motility of sperm obtained from freeze-thawed nocturnal emission were decreased compared with sperm from electroejaculation. Fertilization rates after ICSI using the nocturnal emission sperm were relatively low (45%). One clinical pregnancy was achieved after intrauterine insemination. CONCLUSION(S): The quality of sperm from nocturnal emissions is variable, but it can be used in assisted reproduction procedures to avoid aggressive procedures such as electroejaculation or testis biopsy.

Diagnostic laparoscopy in primary and secondary infertility.

PURPOSE: Our purpose was to compare the diagnostic efficacy of laparoscopy between primary and secondary infertility and to define a subgroup in which the positive findings are low. METHODS: A retrospective review of the cases of 206 infertile women who underwent laparoscopy was conducted. RESULTS: Eighty-two (39.8%) patients were found to have evidence of pelvic disease--20 (22.2%) with primary infertility and 62 (54.3%) with secondary infertility. The ratio of positive findings in secondary infertility was significant in comparison with the positive findings in primary infertility. In only 3 of the 20 patients with primary infertility was there no history of an abdominal operation, pelvic inflammatory disease, or an abnormal hysterosalpingography. Of the 62 women who suffered secondary infertility and had positive findings in laparoscopy, 15 had no history of suspect findings. CONCLUSIONS: The diagnostic yield of laparoscopy for primary infertility in the absence of indications of mechanical factors is low. Therefore the need for diagnostic laparoscopy in these cases should be reconsidered.

Antisperm antibodies in men with psychogenic anejaculation.

Antisperm autoantibodies were determined in 16 men suffering from psychogenic anejaculation who underwent assisted reproduction treatments. Blood and semen samples were collected after transrectal electroejaculation and antisperm antibodies in serum and on the surface of motile spermatozoa were measured using the direct and indirect immunobead binding test. Five men (31%) were found positive for antisperm antibodies. The majority of antibodies were directed against the sperm heads. Surface antibodies were mainly IgA isotype whereas serum antibodies were IgG isotype. These results suggest that psychogenic anejaculation might be associated with increased incidence of antisperm autoimmunity.

Electroejaculation in combination with intracytoplasmic sperm injection in patients with psychogenic anejaculation results in lower fertilization rates.

OBJECTIVE: To evaluate the outcome of intracytoplasmic sperm injection (ICSI) with sperm obtained by electroejaculation in men with psychogenic anejaculation. DESIGN: Retrospective clinical study. SETTING: In Vitro Fertilization Unit, Bikur Cholim Hospital, Jerusalem, Israel. PATIENT(S): Seven men with psychogenic anejaculation who underwent 16 sessions of electroejaculation in combination with ICSI. INTERVENTION(S): Electroejaculation, ICSI. MAIN OUTCOME MEASURE(S): Semen analysis, ICSI, fertilization rates. RESULT(S): All patients had poor sperm motility. One hundred forty-seven oocytes were injected, with a fertilization rate of 27% (39/142). One ongoing pregnancy was achieved. CONCLUSION(S): Sperm obtained by electroejaculation have low motility and reduced fertilization potential. Nevertheless, ICSI should be offered to improve the possibility of successful pregnancy.

Electroejaculation and assisted fertility in men with psychogenic anejaculation.

OBJECTIVES: To evaluate sperm characteristics and fertility potential in ejaculates obtained after electroejaculation in men with psychogenic anejaculation. DESIGN: Retrospective clinical study. SETTING: In Vitro Fertilization Unit, Bikur Cholim Hospital, Jerusalem, Israel. PATIENTS: Twenty men with psychogenic anejaculation who underwent 55 sessions of electroejaculation and their spouses. INTERVENTIONS: Electroejaculation, assisted reproduction technologies. MAIN OUTCOME MEASURES: Semen analysis, IVF, intracytoplasmic injection (ICSI), fertilization rates, and pregnancy rates. RESULTS: In all patients, sperm density and motility rates were unsatisfactory (98 +/- 127 x 10(6) with 14.6% +/- 15% motility in the antegrade portions and 42 +/- 42 x 10(6) with 9.7% +/- 15.6% motility in the retrograde samples). Intrauterine inseminations performed in eight couples did not result in a pregnancy. Four couples underwent IVF-ET treatments. Two pregnancies were achieved with overall success rates of 22% per cycle. Five couples were treated using the ICSI procedure. Although good quality embryos were transferred, none of the treatments resulted in a pregnancy. CONCLUSIONS: Psychogenic failure to ejaculate may be treated by electroejaculation. However, the average motility of the sperm obtained is diminished. The combination of electroejaculation with IVF, including the ICSI procedure, should improve chances of fertilization and pregnancy in these cases.