Improving the performance and evaluation of computer-assisted semen analysis

Semen analysis is performed routinely in fertility clinics to analyze the quality of semen and sperm cells of male patients. The analysis is typically performed by trained technicians or by Computer-Assisted Semen Analysis (CASA) systems. Manual semen analysis performed by technicians is subjective, time-consuming, and laborious, and yet most fertility clinics perform semen analysis in this manner. CASA systems, which are designed to perform the same tasks automatically, have a considerable market share, yet many studies still express concerns about their accuracy and consistency. In this dissertation, the focus is on detection, tracking, and classification of sperm cells in semen images, key elements of CASA systems. The objective is to improve existing CASA algorithms and systems by applying validated computer vision, tracking, and computational intelligence algorithms. The first step of the study is the development of simulation models for generating synthetic images of semen samples. The images enable the assessment of CASA systems and their algorithms. Specifically, the simulation models generate time-lapse images of semen samples for various sperm image categories and include ground truth labels. The models exploit standard image processing operations such as point spread functions and 2D convolutions, as well as new models of sperm cell swimming, developed for this study. They embody multiple studies of sperm motility in the form of parameterized motion equations. Use cases are presented to use the swimming models and the simulated images to assess and compare algorithms for sperm cell segmentation, localization, and tracking. Second, a digital washing algorithm is presented for unwashed semen samples. Digital washing has the potential to replace the chemical washing techniques used by fertility clinics at present, which are costly, time-consuming, and unfriendly to the environment. The digital washing algorithm extracts features from moving sperm cells in an image, and uses these features to identify all sperm cells (moving and stationary) within each studied image (simulated or real). The effectiveness of the digital washing algorithm is demonstrated by comparing the performance of the proposed algorithm to other cell segmentation and detection techniques. Third, a classification algorithm for sperm cells is developed, based on their swimming patterns. The classification algorithm uses K-means clustering on a subset of motility parameters of sperm cells selected by the Artificial Bee Colony (ABC) algorithm. Results of classification and clustering are shown, using simulated and real semen images. Swimming pattern classification has the potential to increase understanding of the relationship between the distribution of sperm cell swimming modes in a patient’s semen image and the fertility of that patient. Lastly, a new method is presented to calculate motility parameters from sperm tracks. The movement of sperm cell is modeled as a sinusoidal traveling wave (“traveling sinusoid”). The amplitude and average path of a moving cell are estimated using an extended Kalman filter (EKF). The states estimated by the EKF include position, velocity, amplitude, and frequency of the traveling wave. The motility parameters calculated from this approach are shown to be superior to those calculated by other existing methods in terms of their accuracy and consistency. CASA developers will find in this study (and in the software made available) new tools to improve the performance of their designs, and to compare and contrast different proposed approaches and algorithms

Counting the Number of Active Spermatozoa Movements Using Improvement Adaptive Background Learning Algorithm

The most important early stage in sperm infertility research is the detection of sperm objects. The success rate in separating sperm objects from semen fluid has an important role for further analysis. This research performed the detection and calculation of human spermatozoa. The detected sperm was the moving sperm in the video data. An improvement of Adaptive Background Learning was applied to detect the moving sperm. The purpose of this method is to improve the performance of Adaptive Background Learning algorithm in background subtraction process to detect and calculate moving sperm on the microscopic video of sperm fluid. This paper also compared several other background subtraction algorithms to conclude the appropriate background subtraction algorithm for sperm detection and sperm counting. The process done in this research was preprocessing using the Gaussian filter. The next was background subtraction process, followed by morphology operation. To test or validate the detection results of any background subtraction algorithm used, the foreground mask results from the morphological operation were compared to the ground truth of moving sperm image. For visualization purposes, every BLOB area (white object in binary image) on the foreground were given a bounding box to the original frame and the number of BLOB objects present in the foreground mask were counted. This shows that the system had been able to detect and calculate moving sperm. Based on the test results, Adaptive Background Learning method had a value of F-measure of 0.9205 and succeeded in extracting sperm shape close to the original form compared to other methods

Advanced methods in reproductive medicine: Application of optical nanoscopy, artificial intelligence-assisted quantitative phase microscopy and mitochondrial DNA copy numbers to assess human sperm cells

Declined fertility rate and population is a matter of serious concern, especially in the developed nations. Assisted Reproductive Technologies (ART), including in vitro fertilization (IVF), have provided great hope for infertility treatment and maintaining population growth and social structure. With the help of ART, more than 8 million babies have already been born so far. Despite the worldwide expansion of ART, there is a number of open questions on the IVF success rates. Male factors for infertility contribute equally as female factors, however, male infertility is primarily focused on the “semen quality”. Therefore, the search of new semen parameters for male fertility evaluation and the exploration of the optimal method of sperm selection in IVF have been included among the top 10 research priorities for male infertility and medically assisted reproduction. The development of imaging systems coupled with image processing by Artificial Intelligence (AI) could be the revolutionary step for semen quality analysis and sperm cell selection in IVF procedures. For this work, we applied optical nanoscopy technology for the analysis of human spermatozoa, i.e., label-based Structured Illumination Microscopy (SIM) and non-invasive Quantitative Phase Microscopy (QPM). The SIM results demonstrated a prominent contrast and resolution enhancement for subcellular structures of living sperm cells, especially for mitochondria-containing midpiece, where features around 100 nm length-scale were resolved. Further, non-labeled QPM combined with machine learning technique revealed the association between gradual progressive motility loss and the morphology changes of the sperm head after external exposure to various concentrations of hydrogen peroxide. Moreover, to recognize healthy and stress-affected sperm cells, we applied Deep Neural Networks (DNNs) to QPM images achieving an accuracy of 85.6% on a dataset of 10,163 interferometric images of sperm cells. Additionally, we summarized the evidence from published literature regarding the association between mitochondrial DNA copy numbers (mtDNAcn) and semen quality. To conclude, we set up the high-resolution imaging of living human sperm cells with a remarkable level of subcellular structural details provided by SIM. Next, the morphological changes of sperm heads resulting from peroxidation have been revealed by QPM, which may not be explored by microscopy currently used in IVF settings. Besides, the implementation of DNNs for QPM image processing appears to be a promising tool in the automated classification and selection of sperm cells during IVF procedures. Moreover, the results of our meta-analysis showed an association of mtDNAcn in human sperm cells and semen quality, which seems to be a relevant sperm parameter for routine clinical practice in male fertility assessment

Sperm cell segmentation in digital micrographs based on convolutional neural networks using u-net architecture

Human infertility is considered a serious disease of the the reproductive system that affects more than 10% of couples worldwide,and more than 30% of reported cases are related to men. The crucial step in evaluating male in fertility is a semen analysis, highly dependent on sperm morphology. However,this analysis is done at the laboratory manually and depends mainly on the doctor’s experience. Besides,it is laborious, and there is also a high degree of interlaboratory variability in the results. This article proposes applying a specialized convolutional neural network architecture (U-Net),which focuses on the segmentation of sperm cells in micrographs to overcome these problems.The results showed high scores for the model segmentation metrics such as precisión (93%), IoU score (86%),and DICE score of 93%. Moreover,we can conclude that U-net architecture turned out to be a good option to carry out the segmentation of sperm cells

Objective evaluation of ram and buck sperm motility by using a novel sperm tracker software

This work offers researchers the first version of an open-source sperm tracker software (Sperm Motility Tracker, V1.0) containing a novel suit of algorithms to analyze sperm motility using ram and buck sperm as models. The computer-assisted semen analysis is used in several publications with increasing trend worldwide in the last years, showing the importance of objective methodologies to evaluate semen quality. However, commercial systems are costly and versatility is constrained. In the proposed method, segmentation is applied and the tracking stage is performed by using individual Kalman filters and a simplified occlusion handling method. The tracking performance in terms of precision (number of true tracks), the percentage of fragmented paths and percentage of correctly detected particles were manually validated by three experts and compared with the performance of a commercial motility analyzer (Microptic's SCA). The precision obtained with our sperm motility tracker was higher than the one obtained with a commercial software at the current acquisition frame rate of 25 fps (P < 0.0001), concomitantly with a similar percentage of fragmentized tracks (P = 0.0709) at sperm concentrations ranging 25-37 106 cells/mL. Moreover, our tracker was able to detect trajectories that were unseen by SCA. Kinetic values obtained by using both methods were contrasted. The higher values found were explained based on the better performance of our sperm tracker to report speed parameters for very fast motile sperm. To standardize results, acquisition conditions are suggested. This open-source sperm tracker software has a good plasticity allowing researchers to upgrade according requirements and to apply the tool for sperm from a variety of species.Fil: Buchelly Imbachí, Francisco Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Pastore, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Greco, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Iniesta-Cuerda, M.. Universidad de Castilla-La Mancha; EspañaFil: Garde, J. J.. Universidad de Castilla-La Mancha; EspañaFil: Soler, A. J.. Universidad de Castilla-La Mancha; EspañaFil: Ballarin, Virginia Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Científicas y Tecnológicas en Electrónica. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones Científicas y Tecnológicas en Electrónica; ArgentinaFil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

High spatially sensitive quantitative phase imaging assisted with deep neural network for classification of human spermatozoa under stressed condition

Sperm cell motility and morphology observed under the bright field microscopy are the only criteria for selecting a particular sperm cell during Intracytoplasmic Sperm Injection (ICSI) procedure of Assisted Reproductive Technology (ART). Several factors such as oxidative stress, cryopreservation, heat, smoking and alcohol consumption, are negatively associated with the quality of sperm cell and fertilization potential due to the changing of subcellular structures and functions which are overlooked. However, bright field imaging contrast is insufficient to distinguish tiniest morphological cell features that might influence the fertilizing ability of sperm cell. We developed a partially spatially coherent digital holographic microscope (PSC-DHM) for quantitative phase imaging (QPI) in order to distinguish normal sperm cells from sperm cells under different stress conditions such as cryopreservation, exposure to hydrogen peroxide and ethanol. Phase maps of total 10,163 sperm cells (2,400 control cells, 2,750 spermatozoa after cryopreservation, 2,515 and 2,498 cells under hydrogen peroxide and ethanol respectively) are reconstructed using the data acquired from the PSC-DHM system. Total of seven feedforward deep neural networks (DNN) are employed for the classification of the phase maps for normal and stress affected sperm cells. When validated against the test dataset, the DNN provided an average sensitivity, specificity and accuracy of 85.5%, 94.7% and 85.6%, respectively. The current QPI + DNN framework is applicable for further improving ICSI procedure and the diagnostic efficiency for the classification of semen quality in regard to their fertilization potential and other biomedical applications in general

Advanced Label-Free Optical Methods for Spermatozoa Quality Assessment and Selection

Current in vitro fertilization (IVF) techniques require a severe selection of sperm, generally based on concentration, morphology, motility, and DNA integrity. Since routinely separation methods may damage the viability of the sperm cell, there is a growing interest in providing a method for noninvasively analyzing spermatozoa taking into account all those parameters. This chapter first reviews the state-of-the-art of label-free sperm cell imaging for IVF, highlighting the limitations of the used techniques. Then, our innovative approach combining Raman spectroscopy and digital holography will be described and its advantages detailed. These include the ability to perform a simultaneous and correlative morphological and biochemical analysis of sperm cells, without labeling, in a fast and reliable way. Finally, the difficulty in reaching clinical use will be discussed, as well as the possible solutions offered by new technological improvements

Image Processing Methods for Automatic in-vitro Morphology Analysis

The study of male infertility has become a priority for biologists and researchers in the last decades as a consequence of the declining birth rates. This problem has also become a major public health with economic and psychosocial consequences. Analysis of human sperm cells, for instance, is widely used in investigations related to male infertility and assisted conception. Sperm samples are usually analysed by health professionals using microscope devices following a manual process to count and describe their morphology. Nevertheless, this practice is prone to errors and time consuming. This thesis proposes a novel framework based on image processing and machine learning methods to automate the analysis of sperm cells. The proposed method presented an average accuracy performance of 96.4% classify automatically sperm cells in three classes: normal, abnormal and non sperm cell. Performance results have been obtained in challenging conditions: presence of uneven illumination, unwanted noise and blurring caused by the focus drift and occlusion of objects as a result of the overlapping of sperm cells, among others. The object of interest, sperm cells, captured in the images used in this research did not receive any staining or fixation treatment prior to their capture. A novel and robust methodology based on deep neural learning is developed as part of the automatic feature selection prior to the classification. Also, video and image database of sperm samples was produced at the Andrology laboratory of the University of Sheffield as part of this work. The database was used to validate the proposed framework for the segmentation and classification of in-vitro cells

A Computer Science Approach to Identify and Classify Hyperactivated Spermatozoa

Effective Assisted Reproductive Technology (ART) relies in part upon accurate but easily conducted measurements of sperm motion parameters. Several established methods are widely used to assess possible reasons for male infertility, in human and veterinary Andrology clinics. Computer-assisted sperm analysis (CASA) devices quantitatively assess sperm motion parameters, which have been defined by the World Health Organization, and include the percentage of motile cells in a sample and the motion characteristics of individual cells, such as curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL). However, CASA analyses fail to define hyperactive sperm motility or determine the prevalence of hyperactively motile sperm in the sample. Hyperactively motile sperm swim in an erratic pattern, and this occurs only at the very end of sperm capacitation, a series of biochemical changes occurring in a sperm which enables it to fertilize an oocyte. The computational challenge for detecting hyperactivated sperm motility lies in precisely modeling sperm movement changes that accurately reflect the sperm\u27s biomedical function, by developing an algorithm that detects and classifies these unique motility patterns. Currently, no such algorithms reliably classify hyperactivated spermatozoa. Therefore, several methods to automatically identify and classify hyperactivated spermatozoa trajectories are described and their performance compared to \u27the gold standard\u27 of visual classification, by experts. The methods considered were: two existing methods, a mathematical modification to one of these, and three new methods, each examined independently and then two were combined to produce an integrated approach. Evaluation of each method was performed by using each to analyze an initial data set containing tracks of hyperactivated and progressive sperm, which had been classified by experts in the field, and then to analyze data sets obtained from actual laboratory samples. Classifications as well as misclassifications were recorded in diffusion matrices. Two methods, the Minimum Bounding Square Ratio (MBSR) and the Rotated Rectangular Linearity (RRL) were more effective in accurately detecting hyperactivated sperm and were similar in correctly classifying hyperactivated sperm. However, RRL misclassified twice as many sperm as MBSR. MBSR also outperformed the other methods in correctly classifying progressively motile sperm and sperm exhibiting transitional motility. After developing this algorithm, it was applied to evaluate sperm from a large experiment to determine if sperm treated with different phosphodiesterase inhibitors, used in erectile dysfunction drugs, exhibit sperm motility. The experiment would not have been possible without these new computer algorithms. Taken together, this research demonstrates that newly developed algorithms can be used to identify critically important features of sperm, such as hyperactivity. One algorithm, MBSR may become an important tool improving Assisted Reproductive Technology\u27s succes