Effects of glutathione treatments during sperm washing and in vitro fertilization on the in vitro early development of embryos of Japanese Black cattle.

This study was conducted to examine the effects of adding glutathione (1 mM) to media used for sperm washing and in vitro fertilization (IVF) on the improvement of early development of embryos produced using cryopreserved spermatozoa of the less IVF-competent bull (the one considered unqualified as spermatozoa supplier for the production of bovine blastocysts using IVF). The cryopreserved spermatozoa of this bull were characterized by normal motility and lower ATP content and blastocyst productivity than those of IVF-competent bulls. The addition of glutathione to the sperm washing medium was more effective in improving the productivity of blastocysts and ATP content than the addition of glutathione to the IVF medium or no glutathione addition at all (control). These results suggest that this simple method may be used to improve the potential of cryopreserved spermatozoa of less IVF-competent bulls to fertilize oocytes in vitro and to induce normal embryonic development after fertilization.

Prevention of HIV transmission with sperm washing within fertile serodiscordant couples undergoing non-stimulated intrauterine insemination.

The purpose of this prospective non-randomized study was to study the effectiveness of semen washing followed by intrauterine insemination (IUI) in Human Immune Deficiency Virus (HIV)-discordant couples in which the male partner was infected, in preventing HIV transmission to uninfected partner and offspring. The study was performed in a private assisted reproductive center specialized in couples with infectious diseases and enrolled sixty-nine fertile couples in which male partner tested positive for HIV, seeking for reproductive treatment. Triple sperm washing followed by viral RNA purification and real-time polymerase chain reaction was performed prior to IUI intervention. HIV transmission to female partner and newborns, and clinical pregnancy rate were the main outcome measures. A total of 180 IUI treatment cycles were performed in 69 couples. There were 16 clinical pregnancies (clinical pregnancy rate/cycle 9.0%, clinical pregnancy rate/patient 23.2%), one of which resulted in miscarriage (6.3%). No seroconversion was detected in the 69 women treated with sperm washing followed by IUI or in any of the newborns (tested at birth and at 3 months of age). Sperm washing followed by IUI is a safe and effective treatment option for serodiscordant couples wishing to conceive and to prevent HIV virus transmission to the mothers and newborns.

Rheotaxis-based microfluidic device for selecting sperm from samples infected with a virus.

OBJECTIVE: To investigate whether the presented rheotaxis-based microfluidic device could be used to separate spermatozoa from viruses (i.e., Zika) in the infected semen sample during the selection and washing process. DESIGN: Quantitative and experimental study of the sperm washing/selection process through the microfluidic platform exploiting the positive rheotaxis of sperm. SETTING: None. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Human sperm were purchased from a sperm bank. The raw semen sample was mixed with viruses and loaded into a microfluidic device. Experiments were performed with 2 different flow rates (0 and 25 µL/minute) to investigate the washing efficiency of the device in the sperm selection process. The sperm sample was collected after 45 minutes and analyzed to check whether the collected sample is free of any infections (viruses) after isolation. RESULT(S): Fluorescent microscopy and quantitative polymerase chain reaction-based analysis showed that the sperm selected with the presented rheotaxis-based microfluidic device at the optimal flow rate (25 µL/minute) was free of any viruses. CONCLUSION(S): We have developed a simple, cost-effective microfluidic device that mimics the conditions of the female genital tract while washing out the raw semen efficiently during the selection process for assisted reproductive technology.

Can HIV-positive gay men become parents? How men living with HIV and HIV clinicians talk about the possibility of having children.

It is now established that people living with HIV who have an undetectable viral load and adhere to antiretroviral treatment cannot transmit HIV to their sexual partners. Previous research has shown that 'being undetectable' changes how HIV-positive gay men experience their sex lives. But how does it affect gay men's reproductive behaviours? And what influence does it have on views about parenthood at a time when gay fatherhood has become more socially accepted and publicly visible? Drawing on qualitative interviews with patients and clinicians at four HIV clinics in London, we identify differences in how interviewees talked about the possibility of having children for HIV-positive men. Both groups, unprompted, frequently referred to sperm washing as a method enabling safe conception. However, whereas clinicians talked about sperm washing as an historical technique, which is no longer necessary, patients spoke of it as a current tool. The men rarely mentioned being undetectable as relevant to parenthood and, when prompted, some said that they did not fully understand the mechanics of HIV transmission. Our findings offer new insights into how biomedical knowledge is incorporated into people's understandings of living with HIV, raising important questions about how the meanings of being undetectable are communicated.

Modulation of seminal plasma content in extended semen improves the quality attributes of ram spermatozoa following liquid preservation at 3-5°C.

Seminal plasma (SP) is known to induce motility and capacitation in spermatozoa curtailing their lifespan when preserved. Hence, this study was conducted to examine the effects of removal of SP from sperm surface prior to liquid preservation either by high dilution (1/15) or by washing and the poststorage treatment with SP (15% and 25%, v/v) on the quality attributes of liquid-preserved ram semen. Over the period of storage, the rapid motility (66.0% and 71.1% vs. 58.3%), straightness (87.1% and 82.1% vs. 79.4%), average path velocity (152.3 and 152.0 µm/s vs. 133.3 µm/s) and the straight-line velocity (131.3 and 127.8 µm/s vs. 108.5 µm/s) were significantly (p < 0.05) higher in both the high-dilution and wash groups as compared to the control (1/3 dilution). The functional membrane integrity (82.3% vs. 77.2%) and noncapacitated sperm count (65.0% vs. 58.7%) were also significantly (p < 0.05) higher in the high-dilution and wash groups, respectively, as compared to the control. The poststorage treatment of sperm with SP significantly (p < 0.05) increased the functional membrane integrity (70.1% vs. 53.8%) and most of the motility attributes as compared to the control (without SP). In conclusion, both the removal of SP prior to liquid preservation and poststorage treatment with SP significantly improved the quality attributes of ram spermatozoa.

1H Magnetic Resonance Spectroscopy of live human sperm.

STUDY QUESTION: Can 1H Magnetic Resonance Spectroscopy (MRS) be used to obtain information about the molecules and metabolites in live human spermatozoa? SUMMARY ANSWER: Percoll-based density gradient centrifugation (DGC) followed by a further two washing steps, yielded enough sperm with minimal contamination (<0.01%) from seminal fluid to permit effective MRS which detected significant differences (P < 0.05) in the choline/glycerophosphocholine (GPC), lipid and lactate regions of the 1H MRS spectrum between sperm in the pellet and those from the 40%/80% interface. WHAT IS KNOWN ALREADY: Current methods to examine sperm are either limited in their value (e.g. semen analysis) or are destructive (e.g. immunohistochemistry, sperm DNA testing). A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Sperm suspended in phosphate buffered saline (PBS) at 37°C were examined by 1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet ('80%' sperm) and the 40/80 interface ('40%' sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of '40%' and '80%' sperm. MAIN RESULTS AND THE ROLE OF CHANCE: DGC with two washes minimized the 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination while retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/GPC 1H MRS peak greater than 3:1 signal to noise ratio (SNR) was estimated at ~3 × 106/ml. The choline/GPC and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis (P < 0.0001; n = 20). ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65-0.67, lactate/lipid ROC AUC = 0.86-0.87. LIMITATIONS, REASONS FOR CAUTION: Only 3-4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the '40%' and '80%' sperm. WIDER IMPLICATIONS OF THE FINDINGS: 1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest.

Fertility care interventions should be provided as the first line options for HIV+ serodiscordant couples who desire children in settings with affordable access to care, regardless of their fertility status.

INTRODUCTION: With increasing life expectancy, couples with at least one partner afflicted with HIV are more commonly pursuing the opportunity to have biologic offspring. Currently, there are no universally accepted recommendations regarding first line reproductive treatments for HIV serodiscordant couples lacking a history of infertility. We strongly believe that fertility care intervention should be the first line treatment, when affordably accessible, over natural conception for HIV serodiscordant couples to achieve pregnancy in a safe and efficacious manner. Discussion: In the era of highly active anti-retroviral therapy, in combination with timed intercourse and pre-exposure prophylaxis for the HIV negative partner, some members of the medical community are arguing in favour of natural conception as a means of achieving pregnancy in this patient population. In our opinion, laboratory assisted fertility methods, including intrauterine insemination, in vitro fertilization, and intracytoplasmic sperm injection with semen washing should be the first line treatment recommendation for HIV serodiscordant couples desiring pregnancy for the following reasons: (1) abundance of evidence in the medical literature supporting the safety profile and efficacy of fertility care intervention in couples with HIV; paucity of data addressing safety of natural conception in comparison to fertility intervention techniques (2) unknown public health impact of promoting natural conception as a safe means of achieving pregnancy (3) ethical implications: patients should be offered the available and accessible treatment option posing the lowest possible known risk to the uninfected partner. Conclusions: We believe that physician assisted fertility care, when affordably accessible, should be the treatment of choice over natural conception. While the preliminary data on natural conception in couples using highly active anti-retroviral therapy/pre-exposure prophylaxis/timed intercourse appears promising, we believe that this approach should be limited to patients in resource poor settings where more sophisticated measures do not exist or for patients that simply cannot afford subspecialty care. There are likely to be unknown psychological and behavioural factors impacted by promoting natural conception and diminishing the importance of safe sex practices. Additionally, it is our moral obligation to patients to offer the affordably accessible treatment interventions that pose the least known risk when considering reproductive options.

Effect of single-layer centrifugation or washing on frozen-thawed donkey semen quality: Do they have the same effect regardless of the quality of the sample?

The aims of this study were to determine the sperm quality of frozen-thawed donkey sperm samples after single-layer centrifugation (SLC) using Androcoll-E in comparison to sperm washing or no centrifugation and to determine if the effect on the sperm quality after SLC or sperm washing depends on the quality of the sample. Frozen-thawed sperm samples from Andalusian donkeys were divided into three aliquots, and they were processed using three different techniques after thawing: uncentrifuged diluted control (UDC), sperm washing (SW), and SLC. Afterward, sperm quality index was estimated by integrating all parameters (total and progressive sperm motility, membrane integrity, and DNA fragmentation) in a single value. The relationship between the sperm quality of thawed UDC samples and the effect on sperm parameters in SW and SLC-selected samples was assessed. Sperm quality index was significantly higher (P < 0.001) in SLC (0.8 ± 0.0) samples than that in UDC (0.6 ± 0.0) and SW (0.6 ± 0.0) samples, regardless of the sperm quality index after thawing of the sperm sample. In conclusion, SLC of frozen-thawed donkey spermatozoa using Androcoll-E-Small can be a suitable procedure for selecting frozen-thawed donkey sperm with better quality, in particular in those samples where an improvement in motility is needed.

Comparative study of sperm washing and selection methods after cryopreservation and its influence on sperm subpopulational structure in a bovine model.

The effect of different sperm washing-selection methods on sperm morphometric characteristics as a study to detect differences in the subpopulational structure has been carried out in detail in a bovine model. Cryopreserved sperm samples from 5 bulls were thawed, pooled, and processed by TALP-washing centrifugation method (TWCM), selective Percoll discontinuous density-gradient centrifugation method (PDGM), and self-migration swim-up separation method (SUMM). Live-dead assay (SYBR-14/ethidium homodimer-1), chlortetracycline assay (CTC), and sperm motility were assessed, and aliquots of sperm were processed for automated sperm morphometry analysis (ASMA) simultaneously before (raw thawed sperm used as control, RTS) and after different sperm washing-selection techniques. Deleterious effects of different methods were evident, particularly on sperm membrane integrity (p < 0.05) and capacitation status (p < 0.05). Moreover, each cell was measured for four primary dimensional parameters, and three shape parameters. All sperm morphometric parameters evaluated were analyzed by principal component analysis (PCA) and multivariate clustering analyses. PCA revealed two principal components for each sperm washing or separation method explaining more than the 91% of the variance. The number of subpopulations found was the same for all methods (four) except for PDGM (three). However, irrespective of the number of subpopulations defined by PCA and clustering analyses, the sperm subpopulational structure was found to be different and strongly influenced by the sperm selection procedure due to statistical differences found regarding the sperm biophysical changes induced by each method used (p < 0.001). It is concluded that different sperm washing-selection methods commonly used during IVF process, may lead to alterations in sperm morphometric characteristics, which might explain the different results seen after IVF, since an important influence of these methods on sperm subpopulational structure has been demonstrated.