scholarly journals A microfluidic approach to rapid sperm recovery from heterogeneous cell suspensions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Steven A. Vasilescu ◽  
Shayan Khorsandi ◽  
Lin Ding ◽  
Sajad Razavi Bazaz ◽  
Reza Nosrati ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Sperm Cells ◽  
Motile Sperm ◽  
Testicular Sperm ◽  
Inertial Microfluidics ◽  
Mixed Suspension ◽  
3D Printed ◽  
Sperm Recovery

AbstractThe isolation of sperm cells from background cell populations and debris is an essential step in all assisted reproductive technologies. Conventional techniques for sperm recovery from testicular sperm extractions stagnate at the sample processing stage, where it can take several hours to identify viable sperm from a background of collateral cells such as white bloods cells (WBCs), red blood cells (RBCs), epithelial cells (ECs) and in some cases cancer cells. Manual identification of sperm from contaminating cells and debris is a tedious and time-consuming operation that can be suitably addressed through inertial microfluidics. Microfluidics has proven an effective technology for high-quality sperm selection based on motility. However, motility-based selection methods cannot cater for viable, non-motile sperm often present in testicular or epididymal sperm extractions and aspirations. This study demonstrates the use of a 3D printed inertial microfluidic device for the separation of sperm cells from a mixed suspension of WBCs, RBCs, ECs, and leukemic cancer cells. This technology presents a 36-fold time improvement for the recovery of sperm cells (> 96%) by separating sperm, RBCS, WBCs, ECs and cancer cells into tight bands in less than 5 min. Furthermore, microfluidic processing of sperm has no impact on sperm parameters; vitality, motility, morphology, or DNA fragmentation of sperm. Applying inertial microfluidics for non-motile sperm recovery can greatly improve the current processing procedure of testicular sperm extractions, simplifying the fertility outcomes for severe forms of male infertility that warrant the surgery.

EFFECTIVENESS OF ASSISTED REPRODUCTIVE TREATMENT PROGRAMS DEPENDING ON THE CHARACTERISTICS OF SPERMOGRAM CHANGES

2018 ◽  
Vol 19 (2) ◽  
pp. 82-87 ◽  
Author(s):  
S. I. Gamidov ◽  
R. I. Ovchinnikov ◽  
A. Yu. Popova ◽  
V. V. Polozov ◽  
N. P. Naumov ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Assisted Reproductive Technologies ◽  
Sperm Concentration ◽  
Reproductive Technologies ◽  
Motile Sperm ◽  
Study Objective ◽  
Normal Morphology ◽  
Live Births ◽  
Significant Difference

Introduction. There’re some pathological mechanisms of male fertility disorders that still don’t have proper diagnostic tests. This significantly decreases diagnostic value of a spermogram and makes the problem of evaluation of the characteristics of spermogram changes and their effects on the effectiveness of assisted reproductive technologies (ART) a pressing problem.The study objectiveis to identify the correlation between effectiveness of ART programs and the characteristics of spermogram changes, in particular sperm concentration, motility, and morphology Materials and methods. At the V.I. Kulakov Research Center for Obstetrics, Gynecology and Perinatology in the period from December of 2012 to December of 2016, 10,042 married couples who underwent treatment using ART (2221 – in vitro insemination (IVF), 7821 – IVF with intracytoplasmic sperm injection) were examined.Results. In patients after IVF, the frequency of live births significantly depended on sperm concentration: 28.6 % for concentration above 5 mil/ml and 51.5 % for concentration above 15 mil/ml (p <0.0001). No significant difference was observed for the dependence of the frequency of live births on the number of progressive-motile sperm (grade А): 38.2 % for <5 % and 57.7 % for >15 % (p = 0.11), or on the number of spermatozoa with normal morphology: 50 % for ≥4 % and 45.5 % for <4 % (p = 0,23). In patients after IVF with intracytoplasmic sperm injection, the concentration of spermatozoa, number of progressive-motile sperm (grade А), and number of spermatozoa with normal morphology didn’t affect the frequency of live births in a statistically significant way.Conclusion. Sperm concentration, motility, and morphology can affect the frequency of live births in the IVF program, but statistically significant correlation was observed only for sperm concentration. After IVF with intracytoplasmic sperm injection, only sperm morphology affects the frequency of live births, but not in a statistically significant way. 

scholarly journals The Roles of NO and H2S in Sperm Biology: Recent Advances and New Perspectives

2020 ◽  
Vol 21 (6) ◽  
pp. 2174
Author(s):  
Martin Kadlec ◽  
José Luis Ros-Santaella ◽  
Eliana Pintus
Keyword(s):  
Assisted Reproductive Technologies ◽  
Male Gamete ◽  
Cell Types ◽  
Reproductive Technologies ◽  
Mechanisms Of Action ◽  
Sperm Function ◽  
Sperm Cells ◽  
Cellular Functions ◽  
Recent Advances ◽  
Potential Use

After being historically considered as noxious agents, nitric oxide (NO) and hydrogen sulfide (H2S) are now listed as gasotransmitters, gaseous molecules that play a key role in a variety of cellular functions. Both NO and H2S are endogenously produced, enzymatically or non-enzymatically, and interact with each other in a range of cells and tissues. In spite of the great advances achieved in recent decades in other biological systems, knowledge about H2S function and interactions with NO in sperm biology is in its infancy. Here, we aim to provide an update on the importance of these molecules in the physiology of the male gamete. Special emphasis is given to the most recent advances in the metabolism, mechanisms of action, and effects (both physiological and pathophysiological) of these gasotransmitters. This manuscript also illustrates the physiological implications of NO and H2S observed in other cell types, which might be important for sperm function. The relevance of these gasotransmitters to several signaling pathways within sperm cells highlights their potential use for the improvement and successful application of assisted reproductive technologies.

Cryopreservation and Preparation of Thawed Spermatozoa from Rhesus Macaques (Macaca mulatta) for In Vitro Fertilization

2021 ◽  
Author(s):  
Fernanda M de Carvalho ◽  
Cathy Ramsey ◽  
Carol B Hanna ◽  
Rodrigo del R do Valle ◽  
Marcilio Nichi ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Assisted Reproductive Technologies ◽  
Rhesus Macaques ◽  
Gradient Centrifugation ◽  
Reproductive Technologies ◽  
Slow Freezing ◽  
Motile Sperm ◽  
Vitro Fertilization ◽  
Isolation Methods

Advances in assisted reproductive technologies in rhesus macaques have allowed the development of valuable models of human disease, particularly when combined with recent techniques for gene editing. While the ability to perform in vitro fertilization (IVF) in rhesus macaques is well established, this procedure has not yet been optimized. Specifically, damage to the sperm caused by cryopreservation (cryodamage) may lead to unsuccessful artificial insemination and low fertilization and blastocyst formation rates in vitro. To address this, we systematically assessed 2 cryopreservation methods and 4 recovery methods in the following 3 interdependent experiments: 1) comparing sperm survival after vitrification or slow-freezing; 2) comparing simple wash (SW), density gradient centrifugation (DGC), swim-up (SU), and glass wool filtration (GWF) for removal of cryoprotectants and isolation of motile sperm after thawing; and 3) evaluating the efficacy for IVF of the 2 best methods of isolating thawed sperm. We found that after vitrification, only 1.2 ± 0.3% of thawed sperm were motile, whereas after slow-freezing, 42 ± 5% of thawed sperm were motile. SW was significantly better than all other isolation methods for the recovery of total sperm and for the recovery of sperm with an intact plasma membrane. The isolation methods had no significant differences in the recovery of motile sperm or sperm with progressive motility. However, IVF of ova with sperm recovered by DGC resulted in 5% more embryos and 25% more blastocysts than did IVF with sperm recovered by SW. Although additional studies are required to optimize sperm cryopreservation in rhesus macaques, our study showed that slow-freezing, coupled with DGC, provided the highest efficacy in providing functional sperm for in vitro use.

scholarly journals Progressive Sperm Separation Using Parallelized, High-Throughput, Microchamber-based Microfluidics

2020 ◽  
Author(s):  
Mohammad Yaghoobi ◽  
Morteza Azizi ◽  
Amir Mokhtare ◽  
Alireza Abbaspourrad
Keyword(s):  
High Throughput ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Biological Research ◽  
Motile Sperm ◽  
Microfluidic Platform ◽  
Short Period ◽  
Sperm Migration ◽  
Fundamental Research ◽  
Sperm Separation

AbstractMotility is one of the most important factors in sperm migration toward egg. Therefore, sperm separation based on motility increases the chance of the best sperm selection in the process of infertility treatments. Unfortunately, it is now vastly done by conventional procedures which lack certain delicacy and precision and increase the risk of damage to sperm cells. Microfluidic systems, on the other hand, can sort sperm in a less intrusive way. However, microfluidic techniques have yet to receive widespread adoption in clinical settings, not only due to their relatively cumbersome operation, but also their extremely low outcome, leaving them inefficient in practice. Here we propose a microchamber-based microfluidic platform that can separate progressive motile sperm from nonviable sperm and debris as well as trapped nonprogressive sperm in the microchambers. Our platform is operated in a short period of time (<10 min) with an excellent degree of controllability, without any prior sample preparation. Our results show that the microchambers’ depth does not affect the residence time of motile sperm. Therefore, we are able to inspect high sample volumes (1 mL) within the same time. Furthermore, we maximize the concentration of the collected sperm by tuning the washing medium flow rate above the sperm rheotactic threshold. We foresee that our microfluidic platform may provide a facile solution for high-throughput, robust, and easy-to-modify for collection of progressive sperm needed for assisted reproductive technologies (ARTs).Significance StatementAssisted Reproductive Technologies require efficient, minimally invasive, and fast methods of sperm separation. Centrifugation methods used in clinics and biological research labs, fall short in these aspects as they are low-yield, intrusive to sperm’s DNA, and time consuming. We have developed a microchamber-based microfluidic platform for high-throughput separation of progressive motile sperm from undiluted raw semen samples. The method was further optimized to increase the concentration of collected samples. Higher concentration of collected samples combined with higher motility of the separated sperm compared to those in raw semen, make it a suitable choice in clinical applications, fertility diagnostics, and fundamental research.

MicroRNA profile comparison of testicular tissues derived from successful and unsuccessful microdissection testicular sperm extraction retrieval in non-obstructive azoospermia patients

2019 ◽  
Vol 31 (4) ◽  
pp. 671 ◽  
Author(s):  
Na Fang ◽  
Congcong Cao ◽  
Yujiao Wen ◽  
Xiaoli Wang ◽  
Shuiqiao Yuan ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Predictive Biomarkers ◽  
Reproductive Technologies ◽  
Small Rna Sequencing ◽  
Testicular Sperm Extraction ◽  
Obstructive Azoospermia ◽  
Testicular Sperm ◽  
Sperm Retrieval ◽  
Altered Expression ◽  
Pathway Analyses

Non-obstructive azoospermia (NOA) is the most severe clinical diagnosis in cases of male infertility. Although in some cases of NOA spermatozoa can be retrieved by microdissection testicular sperm extraction (micro-TESE) to fertilise eggs through intracytoplasmic sperm injection (ICSI), there remains a lack of potential biomarkers for non-invasive diagnosis before micro-TESE surgery. To determine predictive biomarkers for successful sperm retrieval before micro-TESE, the aim of this study was to explore whether microRNAs (miRNAs) were differentially expressed in testicular tissues in NOA patients in whom sperm retrieval had been successful (SSR) versus those in whom it had been unsuccessful (USR) using next-generation small RNA sequencing (RNA-Seq). In all, 180 miRNAs were identified with significantly altered expression levels between SSR and USR testicular tissues. Of these, the expression of 13 miRNAs was upregulated and that of 167 miRNAs was downregulated in the USR compared with SSR group. Unexpectedly, 86 testicular miRNAs were found to be completely absent in the USR group, but showed high expression in the SSR group, suggesting that these miRNAs may serve as biomarkers for micro-TESE and may also play an essential role in spermatogenesis. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that the miRNAs that differed significantly between the USR and SSR groups were involved in cell apoptosis, proliferation and differentiation, which are of considerable importance during spermatogenesis. In summary, this study identified a panel of miRNAs highly expressed in testicular tissues of SSR but not USR NOA patients, providing new insights into specific miRNAs that may play important roles in epigenetic regulation during spermatogenesis. The findings provide a basis for further elucidation of the regulatory role of miRNAs in spermatogenesis and clues to identifying useful biomarkers to predict residual spermatogenic loci in NOA patients during treatment with assisted reproductive technologies.

scholarly journals Corrigendum to: Efficacy of short-term cold storage prior to cryopreservation of spermatozoa in a threatened lizard

2021 ◽  
Vol 33 (9) ◽  
pp. 619
Author(s):  
Lachlan Campbell ◽  
John Clulow ◽  
Belinda Howe ◽  
Rose Upton ◽  
Sean Doody ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Significant Role ◽  
Cold Storage ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Motile Sperm ◽  
Sperm Viability ◽  
Short Term ◽  
Significant Difference

Assisted reproductive technologies (ARTs) have a significant role to play in reptile conservation, yet are severely lacking. Previous attempts to cryopreserve spermatozoa in the threatened lizard Varanus panoptes achieved approximately 48% motile sperm post-thaw for samples frozen immediately after collection. However, the feasibility of extended cold storage before cryopreservation has not been tested. We held V. panoptes spermatozoa at either 25°C or 4°C for 8 days, assessing sperm motility at days 1, 2, 4 and 8. Subsamples were cryopreserved on days 1 and 4 following the previously reported protocol for this species. Percentage motility decreased rapidly at 25°C, but did not decrease significantly until 4 days after collection at 4°C, with &gt;30% motility maintained after 8 days. There was no significant difference in post-thaw motility or viability of samples cryopreserved after 1 or 4 days storage at 4°C, yielding substantial results for both parameters (mean motility 23.8% and 28.1% and mean viability 50.1% and 57.5% after 1 and 4 days respectively). We demonstrate the capacity to extend sperm viability for up to 8 days in unfrozen samples and to produce acceptable post-thaw motility in samples frozen after 4 days of storage, contributing to the development of valuable ARTs for lizards and other reptiles.

scholarly journals Reproductive outcomes predicted by phase imaging with computational specificity of spermatozoon ultrastructure

2020 ◽  
Vol 117 (31) ◽  
pp. 18302-18309 ◽  
Author(s):  
Mikhail E. Kandel ◽  
Marcello Rubessa ◽  
Yuchen R. He ◽  
Sierra Schreiber ◽  
Sasha Meyers ◽  
...  
Keyword(s):  
Cell Viability ◽  
Contrast Agents ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Dry Mass ◽  
Sperm Cells ◽  
Phase Imaging ◽  
Label Free ◽  
Mass Content

The ability to evaluate sperm at the microscopic level, at high-throughput, would be useful for assisted reproductive technologies (ARTs), as it can allow specific selection of sperm cells for in vitro fertilization (IVF). The tradeoff between intrinsic imaging and external contrast agents is particularly acute in reproductive medicine. The use of fluorescence labels has enabled new cell-sorting strategies and given new insights into developmental biology. Nevertheless, using extrinsic contrast agents is often too invasive for routine clinical operation. Raising questions about cell viability, especially for single-cell selection, clinicians prefer intrinsic contrast in the form of phase-contrast, differential-interference contrast, or Hoffman modulation contrast. While such instruments are nondestructive, the resulting image suffers from a lack of specificity. In this work, we provide a template to circumvent the tradeoff between cell viability and specificity by combining high-sensitivity phase imaging with deep learning. In order to introduce specificity to label-free images, we trained a deep-convolutional neural network to perform semantic segmentation on quantitative phase maps. This approach, a form of phase imaging with computational specificity (PICS), allowed us to efficiently analyze thousands of sperm cells and identify correlations between dry-mass content and artificial-reproduction outcomes. Specifically, we found that the dry-mass content ratios between the head, midpiece, and tail of the cells can predict the percentages of success for zygote cleavage and embryo blastocyst formation.

scholarly journals Microfluidic Systems for Isolation of Spermatozoa from Testicular Specimens of Non-Obstructive Azoospermic Men: Does/Can It Improve Sperm Yield?

2021 ◽  
Vol 10 (16) ◽  
pp. 3667
Author(s):  
Gary D. Smith ◽  
Clementina Cantatore ◽  
Dana A. Ohl
Keyword(s):  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Initial Time ◽  
Seminiferous Tubules ◽  
Testicular Sperm Extraction ◽  
Obstructive Azoospermia ◽  
Testicular Sperm ◽  
Laboratory Techniques ◽  
Sperm Aspiration ◽  
Testicular Sperm Aspiration

Intracytoplasmic sperm injection (ICSI) has allowed reproduction options through assisted reproductive technologies (ARTs) for men with no spermatozoa within the ejaculate (azoospermia). In men with non-obstructive azoospermia (NOA), the options for spermatozoa retrieval are testicular sperm extraction (TESE), testicular sperm aspiration (TESA), or micro-surgical sperm extraction (microTESE). At the initial time of spermatozoa removal from the testis, spermatozoa are immobile. Independent of the means of spermatozoa retrieval, the subsequent steps of removing spermatozoa from seminiferous tubules, determining spermatozoa viability, identifying enough spermatozoa for oocyte injections, and isolating viable spermatozoa for injection are currently performed manually by laboratory microscopic dissection and collection. These laboratory techniques are highly labor-intensive, with yield unknown, have an unpredictable efficiency and/or success rate, and are subject to inter-laboratory personnel and intra-laboratory variability. Here, we consider the potential utility, benefits, and shortcomings of developing technologies such as motility induction/stimulants, microfluidics, dielectrophoresis, and cell sorting as andrological laboratory add-ons to reduce the technical burdens and variabilities in viable spermatozoa isolation from testicular samples in men with NOA.

Ultrastructure of Testicular Spermatozoon of the Fish Oreochromis Niloticus

1988 ◽  
Vol 46 ◽  
pp. 278-279
Author(s):  
T. Guha ◽  
A. Q. Siddiqui ◽  
P. F. Prentis
Keyword(s):  
Electron Microscope ◽  
Oreochromis Niloticus ◽  
Osmium Tetroxide ◽  
Sperm Cells ◽  
Adult Fish ◽  
Testicular Sperm ◽  
Thin Sections ◽  
Important Fish ◽  
Testicular Spermatozoon ◽  
Diamond Knife

Tilapia, Oreochromis niloticus, is an economically important fish in Saudi Arabia. Elucidation of reproductive biology of this species is necessary for successful breeding program. In this paper we describe fine structure of testicular sperm cells in O, niloticus.Testes from young adult fish were fixed in gluteraldehyde (2%) and osmium tetroxide (1%), both in cacodyl ate buffer. Specimens were processed in the conventional way for electron microscopy and thin sections of tissues (obtained by cutting the blocks with a diamond knife) were stained by ura- nyl acetate and lead citrate. These were examined in a Carl Zeiss electron microscope operated at 40 kV to 60 kV. Sperm cells were obtained from testes by squeezing them in cacodyl ate buffer. They were fixed in gluteraldehyde (2%) in the same buffer, air dried, gold coated and then examined in a Philips scanning electron microscope (SEM) operated at 25kV.The spermatozoon of O. niloticus is consisting of head, midpiece and tail (Fig. 1).

Re-imagining Reproduction: Unsettling Metaphors in the History of Imperial Science and Commercial Surrogacy in India

Somatechnics ◽  
2015 ◽  
Vol 5 (1) ◽  
pp. 88-103 ◽  
Author(s):  
Kalindi Vora
Keyword(s):  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
The Body ◽  
Body Parts ◽  
Colonial History ◽  
Women's Bodies ◽  
Commercial Surrogacy ◽  
Art Practices ◽  
History Of ◽  
Travel Industry

This paper provides an analysis of how cultural notions of the body and kinship conveyed through Western medical technologies and practices in Assisted Reproductive Technologies (ART) bring together India's colonial history and its economic development through outsourcing, globalisation and instrumentalised notions of the reproductive body in transnational commercial surrogacy. Essential to this industry is the concept of the disembodied uterus that has arisen in scientific and medical practice, which allows for the logic of the ‘gestational carrier’ as a functional role in ART practices, and therefore in transnational medical fertility travel to India. Highlighting the instrumentalisation of the uterus as an alienable component of a body and subject – and therefore of women's bodies in surrogacy – helps elucidate some of the material and political stakes that accompany the growth of the fertility travel industry in India, where histories of privilege and difference converge. I conclude that the metaphors we use to structure our understanding of bodies and body parts impact how we imagine appropriate roles for people and their bodies in ways that are still deeply entangled with imperial histories of science, and these histories shape the contemporary disparities found in access to medical and legal protections among participants in transnational surrogacy arrangements.

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