Selection Of Motile Sperm Research Articles

Computational and in vitro binding studies of the redesigned pentoxifylline analogue against phosphodiesterases function in sperm

Pentoxifylline (PTX) is a widely used pharmacological agent for the selection of motile sperm in both normozoospermic and asthenozoospermic ejaculates prior to their use in assisted reproductive technologies (ARTs), e.g. intracytoplasmic sperm injection (ICSI). Since cAMP signalling pathways underlie motility, PTX stimulates motility through the inhibition of cAMP-hydrolysing phosphodiesterase (PDE) isoforms in sperm, viz. PDE4A, PDE4D, and PDE10A. However, it has been noted that PTX causes an early acrosome reaction (AR) and has deleterious effects on oocyte development and early embryogenesis. Previous in vitro and in silico binding studies on the aforementioned PDEs with PTX and a modified PTX called PTXm-1 suggested room for improvement in terms of binding affinity. Indeed, using a structure-guided in silico approach we designed a novel modified PTX (mPTX), which outperformed both PTX and PTXm-1 in binding to PDEs in our in silico binding studies based on docking and molecular dynamics (MD) as well as in vitro binding studies employing isothermal titration calorimetry (ITC). Our findings warrant comparative ex vivo evaluation of mPTX to know whether mPTX is more effective than PTX and PTXm-1 vis-à-vis functional competence for assisted reproductive technology.

High-throughput selection of sperm with improved DNA integrity and rapidly progressive motility using a butterfly-shaped chip compared to the swim-up method.

Microfluidics provides unique opportunities for the high throughput selection of motile sperm with improved DNA integrity for assisted reproductive technologies (ARTs). Here, through a parametric study on dimensions and geometrical angles, a butterfly-shaped chip (BSC) is presented to isolate sperm with high progressive motility and intact DNA at a separation rate of 1125 sperm per minute. Using finite element simulations, the flow field and shear rates in the device were optimized to leverage the inherent motility characteristics of sperm for maximum selection throughput. The device incorporates a triple selection mechanism in series, initially activating sperm rheotaxis by rotation against the semen flow, penetrating the counter buffer flow and swimming against the direction of the buffer flow, leaving dead cells and debris behind, and subsequently leveraging boundary-following behavior to direct progressively motile sperm to swim along the walls and reach the device outlet. The device selects over 4.1 million sperm per mL within 20 minutes, with 29.2%, 68.2%, and 57.3% improvement in total motility, DNA integrity, and velocity parameter (VCL), as compared with the conventional swim-up method, respectively. Overall, the performance of the device to separate sperm with approximately 95.9% total motility, 97.8% viability, and 96.6% DNA integrity at high concentrations demonstrates its potential for enhancing the efficiency of conventional treatment methods.

P-084 Novel Single-Step Microfluidic Chamber Outperforms Density Gradient Centrifugation in Post-Selection Motility And DNA Integrity

Abstract Study question Does sperm selection by a novel single-step microfluidic chamber offer effective selection of motile sperm without DNA damage compared with Density Gradient Centrifugation (DGC)? Summary answer The novel single-step microfluidic chamber is a good alternative to conventional DGC, increasing DNA integrity and progressive sperm motility in recovered sperm populations. What is known already High levels of sperm DNA fragmentation (SDF) can have negative impacts on reproductive outcomes such as clinical pregnancy and miscarriage rates. Microfluidic sperm selection technologies have been developed to select sperm with lower level of SDF and higher levels of motility to improve Assisted Reproductive technology (ART) outcomes. While these methods have achieved variable success, very few have proven clinically relevant and fail to perform selection in a robust and simple methodology that can be adopted in clinics. Study design, size, duration A prospective cohort study including 21 donated semen samples processed in 2021. Each sample was split to perform DGC and microfluidic sperm selections side-by-side. Laboratory outcomes were evaluated using semen parameters pre and post processing and included concentration, motility, and DNA fragmentation index (DFI). Participants/materials, setting, methods The study was performed using samples donated at the Department of Obstetrics and Gynaecology, Monash University, Melbourne, Australia. DGC (10 minutes at 300g and 5 minutes wash at 300g) and microfluidic sperm selection (one-step sperm selection;15-minute incubation for selection) were performed with Sydney IVF 80/40 gradients and gamete buffers, respectively. DFI was assessed by chromatin sperm dispersion assay (SCD). Results were analysed by Friedman’s test and differences were considered significant when p-value < 0.05. Main results and the role of chance For the unprocessed raw semen, the starting DFI values varied from 3.0 to 23.3% with an average of 11.38% ± 5.51 and motility varied between 25.3% and 86.7% with an average of 56.42% ± 18.57. The device consistently outperformed DGC in all 21 samples for DFI (0.94% ± 1.12 vs 4.87% ±3.69, P = 0.0012) and in 20 of the 21 samples for motility post-processing DGC (90.88% ± 6.8 vs 73.58% ± 14.97, P = 0.0061). The average concentrations of the raw, DGC, and microfluidic sperm samples were 94.5, 74.12, and 3.55 million sperm/mL, respectively. Thus, both DGC and the microfluidic device showed improvements in SDF, motility, and concentration over the raw sample. However, the microfluidic device significantly outperformed DGC in terms of percentage SDF and motility, indicating its potential for clinical implementation. Limitations, reasons for caution This cohort study did not include samples from infertile patients. Furthermore, the effect of each sperm selection methods on reproductive outcomes was not a part of the study. A larger number of samples across a range of clinically infertile samples is required to fully characterise the microfluidic devices efficacy. Wider implications of the findings The microfluidic device we report here has the potential to become a new tool to improve the efficacy and consistency of sperm selections. When compared to DGC it offers improved sperm motility and DNA integrity from a platform that is simple to use and less skill-based than conventional methods. Trial registration number not applicable

A PMMA-Based Microfluidic Device for Human Sperm Evaluation and Screening on Swimming Capability and Swimming Persistence.

The selection of high-quality sperm is essential to the success of in vitro fertilization (IVF). As human cervical mucus has a high viscosity, without enough swimming persistence, human sperm clouds cannot arrive at the ampulla to fertilize the egg. In this study, we used swimming capability and motion characteristics that are known to be associated with fertilization ability to evaluate the quality of sperm. Here, a clinically applicable polymethyl methacrylate (PMMA)-based microdevice was designed and fabricated for sperm evaluation and screening for swimming capability and persistence in a viscous environment. In this study, we applied methylcellulose (MC) to mimic the natural properties of mucus in vivo to achieve the selection of motile sperm. Sperm motion was recorded by an inverted microscope. The statistical features were extracted and analyzed. Hundreds of sperm in two treated groups with different concentrations of MC and one control group with human tubal fluid (HTF) media were video recorded. This device can achieve a one-step procedure of high-quality sperm selection and achieve the quantitative evaluation of sperm swimming capability and persistence. Sperm with good swimming capability and persistence may be more suitable for fertilization in a viscous environment. This microdevice and methods could be used to guide the evaluation of sperm motility and screening in the future.

Incorporación del test de dispersión de la cromatina espermática al laboratorio andrológico

ObjectiveTo develop the sperm chromatin dispersion test (SCD) for evaluating the DNA fragmentation in semen. Materials and methods57 normozoospermic (WHO 2010) patients were invited to participate in the study after signing the consents. DNA fragmentation was evaluated by SCD, objectively and subjectively, after oxidative stress with H2O2 and also in motile selected samples (swim up or density gradient), as well as in frozen and thawed samples. ResultsThe objective assessment showed that minor fragmentation is associated to a larger halo size by definition, but also a significant increase in the size of the nucleus is observed. Oxidizing treatment (>100μM of H2O2) increases the fragmentation index in a dose-depend way without affecting sperm viability and motility. Different techniques of motile sperm selection (swim up or density gradients) decrease the fragmentation index with an individual susceptibility. Regardless of the sample (semen, suspension or post swim up direct freezing to –20°C allows to delay the test. ConclusionSubjective evaluation of the SCD test is sufficient for the clinical laboratory, evaluating the relation halo/nucleoid for the classification. To set up the SCD a dose-response curve with oxidizing treatment is required as a positive control of damage. The use of sperm selection methods used in assisted reproduction, decreases the fragmentation index, however individual susceptibility was observed. The SCD could be delayed by freezing the sample which allows saving of reagents and processing time.

Retrospectiva de la tecnología de laboratorio en reproducción asistida

Se revisa la secuencia evolutiva de la tecnología de laboratorio en reproducción asistida. Desde los primeros intentos de manipular embriones preimplantacionales recuperados de oviductos animales, en 1912, las transferencias directas o con breves periodos de incubación a madres receptoras, el complejo proceso bioquímico de preparación y suplementación de los medios de cultivo a partir de soluciones salinas químicamente definidas, la etapa de transición de esta tecnología a los humanos en la que se trabajo básicamente la maduración de ovocitos y la selección de espermatozoides motiles in vitro, hasta los procedimientos que culminaron con la primera aplicación clínica descrita en 1980.

High-throughput microfuidic device for motile sperm selection

Current techniques of semen processing and sperm selection used in preparation for IVF are crude and differ greatly from the natural in vivo processes. Recent studies have shown that microfluidic channels and microfluidic chips may be well-suited for sperm selection. Our aim is to develop and test new microfluidic devices in their ability to separate viable, highly motile sperm with high DNA integrity from non-motile sperm and debris.

Influence of human sperm motility and cryopreservation on the oocyte activating factor phospholipase C zeta

Absence, abnormal expression or defective forms of the oocyte activating factor phospholipase C zeta (PLCz) are linked to failed fertilization. This study addressed the effect of motile sperm selection and cryopreservation on PLCz localization.

When ONE is the not-so-loneliest number!

When ONE is the not-so-loneliest number!

In Vitro Fertilization on a Single-Oocyte Positioning System Integrated with Motile Sperm Selection and Early Embryo Development

In vitro fertilization (IVF) technology has been broadly applied to solve human infertility in recent years. However, the physical tools for IVF remain unchanged over several decades before microfluidic technology was introduced in this field. Here, we report a novel microdevice that integrates each step of IVF, including oocyte positioning, sperm screening, fertilization, medium replacement, and embryo culture. Oocytes can be singly positioned in a 4 × 4 array of octacolumn units. The four symmetrical straight channels, crossing at the oocyte positioning region, allowed efficient motile sperm selection and facilitated rapid medium replacement. The fertilization process and early embryonic development of the individual zygote was traced with microscopic recording and analyzed by in situ fluorescent staining. The murine sperm motility was increased from 60.8 ± 3.4% to 96.1 ± 1.9% through the screening channels. The embryo growth rate and blastocyst formation were similar between the routine Petri dish group and the microdevice group. The healthy blastocysts developed in the microdevice could be conveniently retrieved through a routine pipetting operation and used for further embryo transfer.

14 MOTILITY AND FERTILITY OF BULL SPERMATOZOA FROZEN IN EGG YOLK EXTENDER SUPPLEMENTED WITH LACTOFERRIN

Lactoferrin (Lf) is secreted by the prostate, seminal vesicles, and epididymis, and appears to represent a major component of the sperm-coating antigens that protect sperm. The aim of this study was to determine the effects of Lf used in semen extender on motility and fertility of bovine frozen semen. Semen samples were collected from three fertile Japanese Black sires. The semen collected was cooled and extended with egg yolk–Tris extender supplemented with glycerol and Lf (from bovine milk) to give final concentrations of 7% and 0, 10, 100, 500, 1000, or 2000 �g mL-1, respectively. After being extended, the semen was packaged in 0.5-mL straws, frozen, and transferred into liquid nitrogen. At least one week later, the frozen semen was thawed and washed mTALP containing caffeine. Motile sperm were separated by discontinuous 2-step (40 and 80%) Percoll gradient centrifugation. Sperm motility was calculated as the percentage of motile sperm separated by Percoll gradients relative to pre-separated sperm. Sperm motility character (motile sperm, progressive sperm) was analyzed by a CASA system (Hamilton Thorne Biosciences, Beverly, MA, USA). In the following experiment, we examined the effect of Lf added in the extender on improvement of semen fertility. The frozen semen from one sire extended with or without 500 �g mL-1 of Lf was prepared (99 straws with Lf, 99 straws without), distributed to three inseminators, and used to inseminate to 198 cows. The sperm motility and motility character were analyzed by ANOVA and Fisher's exact test. The field trials were evaluated by chi-square test. After selection of motile sperm with the Percoll gradient, examination of sperm motility revealed that addition of Lf in the extender increased sperm motility significantly (P < 0.05) compared to that in the control without Lf: 51.2–53.8% in semen with added Lf (100–2000 �g mL-1) vs. 39.6% in the control. CASA analysis showed that motile sperm and progressive sperm were improved significantly by the addition of Lf; the peak level was 500 �g mL-1 of Lf in the extender. The percentage of motile sperm in semen with added Lf was higher significantly (P < 0.01) than that of the control: with Lf of 100 and 500 �g mL-1, 50.8 and 56.0%, respectively, vs. 39.7% without Lf. The percentage of progressive sperm was also significantly higher (P < 0.05) than that of the control: with Lf of 100 and 500 �g mL-1, 18.8 and 19.0%, respectively, vs. 13.5% without Lf. In the next experiment, we performed field trials to assess the availability of Lf for artificial insemination. In total, the group with Lf showed some but not a significant increase in fertility rate (delivery cow/inseminated cow) compared with the control group without Lf: 56.6% vs. 52.5%, respectively. In conclusion, these results suggest that addition of Lf in sperm extender may be useful for improvement of sperm motility and motility character after freezing and thawing. In the present study, we used fertile bulls, but the concentration of intact Lf in the semen was unknown, which might be the reason why the effect of Lf on the fertility rate was not observed to be significant. In future experiments it would be valuable to demonstrate the effect of Lf in subfertile semen.

In vitro embryo production through modification of time and gonadotropin hormone during oocytes maturation

This research has been conducted at the laboratory of in vitro fertilization of the University of Wisconsin, USA. These embryos can be used for improving genetic value of Indonesian cattle. There is transportation constraint in importing oocytes from USA. It takes more than 24 hours to bring it to Indonesia. In fact, oocytes maturation and ready to be fertilized normally requires only 24 hours in 5% CO2 incubator at 38.5°C. Therefore, this research is needed to study the effect of gonadotropin hormone and time for oocyte maturity and ready to be fertilized at a period more than 24 hours. If this problem could be solved then the importation of oocytes could be cheaper and easier than importation of life animals or embryos. Ovaries were collected from slaughterhouse in Wisconsin. Oocytes were matured in TCM-199 medium in 5% CO2 incubator and at 30°C enriched with FSH 10 μl/ml, oestradiol 17 β 1μl/ml and 10 % FCS as control of gonadotropin hormone treatment (A); with FSH 10 μl/ml (B); with oestradiol 17 β 1μl/ml (C) and without gonadotropin hormone (D) for 24 hours, 30 hours and 36 hours as time of maturation treatment I, II and III respectively. The oocytes were fertilized in vitro with motile sperm selection by Percoll gradient and incubation between sperm and oocytes in fertilization media (TALP) for 20 hours. All zygotes were cultured in modification of KSOM medium up to blastocyst and were fed serum 5 μl/50 μl medium on day 6. Data were analyzed by SAS program. Percentage of cleavage between time of maturation were significant (p 0.05). Percentage of blastocyst between time of maturation were not significant (p>0.05), but between gonadotropin hormone treatment A vs B and A vs C and B vs D and C vs D were significant (p 0.05). Percentage of cleavage, morula, blastocyst, expanded blastocyst and unertilized ova on this study are 66.73%, 22.43%, 40.33%, 0.81% and 32.51 for 24 hours incubation (I); 61.55%, 25.69%, 32.69%, 0.54% and 27.61% for 30 hours incubation (II); 72.43%, 32.06%, 37.97%, 0.0% and 25.31% for 36 hours incubation (III) respectively. According to this study, in vitro production of embryo could be conducted at 30°C and incubation on maturation media for more than 24 hours. Key words: Oocytes, cleavage, embryos, blastocyst

Cytogenetic studies in motile sperm from normal men.

Most studies on human sperm chromosomes from normal men involve the heterologous fertilization of zona free hamster eggs by unselected human sperm. In this work, we have performed cytogenetic studies of highly motile sperm, selected by a swim-up method. A total of 505 motile human sperm complements from three normal donors was analysed. The total frequency of sperm with chromosomal abnormalities (10.9%; 6.9% structural aberrations and 4.0% aneuploidy) and the sex ratio (50.4% X:49.6% Y) were similar to those obtained from whole semen samples. Our results indicate that the selection of motile sperm does not imply chromosomal selection.

In vitro fertilization outcome: glass wool-filtered sperm versus swim-up sperm

At present, most in vitro fertilization (IVF) programs use a swim-up procedure for sperm preparation. This method mimics the in vivo selection of motile sperm by cervical mucus. Filtration through a column of glass wool, on the other hand, may result in recovery of more viable sperm from an ejaculate with relatively poor quality as compared with the swim-up procedure. Therefore, this study was designed to compare the sperm selected by these two methods, namely the swim-up procedure and the filtration procedure with a standardized glass wool column, with regard to the ability to fertilize human oocytes in our IVF program

Evaluation of Nycodenz and Percoll density gradients for the selection of motile human spermatozoa

Two high-density media, Percoll (Pharmacia, Uppsala, Sweden) and Nycodenz (Nyegaard & Co., Oslo, Norway), were assessed for efficacy of selection of motile spermatozoa from oligozoospermic and asthenozoospermic semen samples. The results indicated initially that the media were equivalent for motile sperm selection. However, only the spermatozoa selected from discontinuous Nycodenz gradients retained their motility for at least 21 hours (60% motility compared with 5% motility). A significantly higher number of motile spermatozoa were harvested from discontinuous Nycodenz gradients than from matched control samples using the swim-up procedure (12.0 +/- 3.0 standard error of the mean [SEM] X 10(6)/ml versus 1.9 +/- 0.7 SEM X 10(6)/ml; 27 experiments, P less than 0.001). Discontinuous Nycodenz gradients may be useful for selection of motile spermatozoa from poor-quality semen for in vitro fertilization.