Silvia W. Lestari

Sperm chromatin maturity and integrity correlated to zygote development in ICSI program

ABSTRACT This study aimed to evaluate sperm chromatin maturity and integrity of that injected into good-quality oocytes in an in vitro fertilization-intra cytoplasmic sperm injection (IVF-ICSI) program. A cut-off value of sperm chromatin maturity and integrity was developed as a function of their correlation to the zygote development, i.e., embryo formation and cleavage rate. The study assessed sperm chromatin maturity using aniline blue (AB) staining, whereas toluidine blue (TB) staining was used to assess sperm chromatin integrity. Ejaculates from 59 patients undergoing ICSI and 46 fertile normozoospermic donors for determination of normal values of sperm chromatin status were used in this study. Embryo formation and cleavage rates were observed for the period of 3 days after ICSI. There was a significant difference in the percentage of sperm with mature chromatin between ejaculate from ICSI patients and fertile donor (p=0.020); while there was no significant difference in sperm chromatin integrity of both samples (p=0.120). There was no significant correlation between sperm chromatin maturity and either embryo formation or cleavage rate; as well as sperm chromatin integrity to both parameters of zygote development (p>0.05). Furthermore, we found that the cut-off value of sperm chromatin maturity and integrity of the fertile normozoospermic ejaculates were 87.2% and 80.2%, respectively. Using the cut-offs, we found that low sperm chromatin maturity at the level of <87% correlated significantly with the cleavage rate of the zygote (p=0.022; r=0.371); whereas poor sperm chromatin integrity at the level of <80% correlated with embryo formation (p=0.048; r=0,485). In conclusion, this study showed that poor maturity and integrity of sperm chromatin (AB<87% and TB<80%, respectively), could affect zygote development following ICSI. Abbreviations: AB: aniline blue; CMA3: chromomycin A3; ICSI: intra cytoplasmic sperm injection; IVF: in vitro fertilization; PBS: phosphate buffer saline; SPSS: Statistical Package for Social Science; TB: toluidine blue; WHO: World Health Organization

Human Sperm tracking using Particle Swarm Optimization combined with Smoothing Stochastic sampling on low frame rate video

In this paper, we present a technique for visual tracking in the field of Human Sperm motion. Application of sperm cell tracking is mainly important in Intracytoplasmic Sperm Injection (ICSI), a medical procedure that has enabled the In Vitro Fertilization (IVF) of a single sperm which is injected directly into an egg. In this paper, we consider the problem of tracking single object in video sequences of human sperms and a newly developed Smoothing Stochastic Approximate Monte Carlo (SSAMC) based tracker enhanced by Particle Swarm Optimization (PSO). The problem for this research is that the motility or movement of Human Sperm is fast and unpredictable. In addition, each and every sperms have closely similar size and shape. To solve this problem, we used PSO for searching algorithm (finding the best target) in a Search Window, it can reduce the search space in every each consecutive frame. The measurement results of the proposed method are then compared with the manual measurements done by experts. The experiment results were conducted on both open video data and our own video data. Experiment results showed that the proposed method can handle our specific problem in human sperm cell tracking, and give us a better result as compared to our previous tracker, which used geometric transition dynamic model and without any enhancement by PSO.

Sperm Na+, K+-ATPase α4 and plasma membrane Ca2+-ATPase (PMCA) 4 regulation in asthenozoospermia

ABSTRACT Asthenozoospermia, which is characterized by reduced motility, is one of the etiologies of male infertility. Its biochemical and functional consequences include altered ATPase activity. This study investigated the activities of Na+, K+-ATPase and Ca2+-ATPase and the expression of Na+, K+-ATPase α4 and PMCA4 isoforms in human sperm of asthenozoospermic infertile men. Nineteen samples from asthenozoospermic infertile couples were examined in this study. Computerized-assisted semen analysis (CASA) was performed, and the enzyme activity was measured based on the ability of ATPase to release organic phosphate from ATP as a substrate. The Na+, K+-ATPase α4 and PMCA4 isoform expression levels were measured by western immunoblotting, whereas the protein distribution was examined by immunocytochemistry. This showed that the Na+, K+-ATPase activity and the Na+, K+-ATPase α4 isoform expression were lower in the asthenozoospermia group than in the normozoospermia group (8.688±1.161 versus 13.851±1.884 µmol Pi/mg protein/h, respectively; p>0.05). In contrast, the Ca2+-ATPase activity was significantly higher in the asthenozoospermia group than in the normozoospermia group (11.154±1.186 versus 2.725±0.545 µmol Pi/mg protein/h, respectively; p<0.05). In comparison, PMCA4 expression in the asthenozoospermia group was lower than in the normozoospermia group (p>0.05). The altered ATPase activity and isoform expression in asthenozoospermia may impair sperm structure and function.

Comparison of sucrose and trehalose media modification as an update of oocyte vitrification: A study of apoptotic level

Number of women who are not being able to have offspring in their reproductive life is increasing which might be influenced by several factors. As a consequence, oocyte cryopreservation could be an ensuring solution for women fertility preservation. A good vitrification could be conducted by combining an appropriate of type and concentration of cryoprotectants. One of the marks of successful vitrification is the vitrified oocytes could avoid apoptosis. This study aimed to evaluate the modification of cryoprotectant media as un update of oocyte vitrification as follow: the combination and the concentration of cryoprotectant media of oocytes vitrification, based on their effects on the apoptosis or DNA damage of oocytes. A total of 84 MII stage oocytes from adult female Deutschland, Denken and Yoken (DDY) mice (7-8 weeks old) were used in this study. Vitrification procedure was performed by using VS1 contained 15% EG, 15% DMSO, 0.5 mol/l sucrose (Merck, Darmstadt, Germany) and VS2 contained 15% EG, 15% DMSO, 0.5 mol/l trehalose (Merck, Darmstadt, Germany) in HM. Furthermore, warming solution (WS) was divided into four groups. There were: WS1a contained 0.3 mol/l sucrose, WS1b contained 0.15 mol/l sucrose, WS2a contained 0.3 mol/l trehalose, and WS2b contained 0.15 mol/l trehalose. Apoptotic level was performed by staining the oocytes with TUNEL and propidium iodide (PI) based on Brison and Schultz method then examined under confocal microscope. The rate of apoptosis in oocytes after vitrification and warming was higher compared to the fresh control oocytes. Furthermore, the rate of apoptosis in the vitrified oocytes by sucrose media (28%) was higher compared to the vitrified oocytes by trehalose media (16%). The results of this study indicated that vitrification increased apoptosis in the vitrified oocytes related to the oocyte injury after vitrification. Moreover, the vitrification increased apoptosis more in the vitrified oocytes by sucrose media than the vitrified oocytes by trehalose media. The exposure to the 16.5% EG, 16.5% DMSO and 0.5 mol/l trehalose as cryoprotectant media decreased their viability and increased the number of DNA-fragmented nuclei in the oocytes, lesser than sucrose. Trehalose was proved to be the more suitable extracellular cryoprotectant media in oocyte vitrification based on the apoptotic level, compared to that of sucrose. A modification of cryoprotectant media as an update of oocyte vitrification consisted 0.5 mol/l trehalose concentration as extracellular cryoprotectant and combined with 16.5% EG and 16.5% DMSO as intracellular cryoprotectant has produced.Number of women who are not being able to have offspring in their reproductive life is increasing which might be influenced by several factors. As a consequence, oocyte cryopreservation could be an ensuring solution for women fertility preservation. A good vitrification could be conducted by combining an appropriate of type and concentration of cryoprotectants. One of the marks of successful vitrification is the vitrified oocytes could avoid apoptosis. This study aimed to evaluate the modification of cryoprotectant media as un update of oocyte vitrification as follow: the combination and the concentration of cryoprotectant media of oocytes vitrification, based on their effects on the apoptosis or DNA damage of oocytes. A total of 84 MII stage oocytes from adult female Deutschland, Denken and Yoken (DDY) mice (7-8 weeks old) were used in this study. Vitrification procedure was performed by using VS1 contained 15% EG, 15% DMSO, 0.5 mol/l sucrose (Merck, Darmstadt, Germany) and VS2 contained 15% EG, 15% DMSO...

Sperm quality after swim up and density gradient centrifugation sperm preparation with supplementation of alpha lipoic acid (ALA): A preliminary study

Intra uterine insemination (IUI) as one of the treatment for infertility, persists low success rate. A factor that contributes to the unsuccessful of IUI is sperm preparation, performed through Swim-up (SU) and Density Gradient Centrifugation (DGC) methods. Furthermore, studies have shown that Alpha Lipoic Acid (ALA) is a potent antioxidant that could enhance the sperm motility and protect the DNA integrity of the sperm [1]. This study is aimed to re-evaluate the efficiency of the DGC and SU methods in selecting sperm before being transferred for IUI by the supplementation of ALA based on the sperm DNA integrity. Semen samples were obtained from 13 men from partners of women who are infertile (normozoospermia) and underwent IUI. Semen analysis based on the guideline of World Health Organization (WHO) 2010 was performed to measure the sperm motility and velocity, before and after sperm preparation. Then, samples were incubated with Alpha Lipoic Acid (ALA) in 0.625 mg (ALA 1), 1.25 mg (ALA 2) and 2.5 mg (ALA 3). The Sperm Chromatin Dispersion (SCD) test was performed to evaluate the sperm DNA Fragmentation Index (DFI). The percentage of motile sperm was higher in prepared sperm (post-DGC and post-SU) than in whole semen. Furthermore, the percentage of motile sperm was higher in post-DGC compared to post-SU. The level of DFI after the supplementation of ALA was decreased in prepared sperm compared to the whole semen. ALA was proved capable to select the better sperm quality with decreased sperm DNA fragmentation of prepared sperm in the all of DFI category.Intra uterine insemination (IUI) as one of the treatment for infertility, persists low success rate. A factor that contributes to the unsuccessful of IUI is sperm preparation, performed through Swim-up (SU) and Density Gradient Centrifugation (DGC) methods. Furthermore, studies have shown that Alpha Lipoic Acid (ALA) is a potent antioxidant that could enhance the sperm motility and protect the DNA integrity of the sperm [1]. This study is aimed to re-evaluate the efficiency of the DGC and SU methods in selecting sperm before being transferred for IUI by the supplementation of ALA based on the sperm DNA integrity. Semen samples were obtained from 13 men from partners of women who are infertile (normozoospermia) and underwent IUI. Semen analysis based on the guideline of World Health Organization (WHO) 2010 was performed to measure the sperm motility and velocity, before and after sperm preparation. Then, samples were incubated with Alpha Lipoic Acid (ALA) in 0.625 mg (ALA 1), 1.25 mg (ALA 2) and 2.5 mg (AL...

Sperm Na+, K+-ATPase and Ca2+-ATPase activity: A preliminary study of comparison of swim up and density gradient centrifugation methods for sperm preparation

As one of the treatment for infertility, the success rate of Intrauterine Insemination (IUI) is still relatively low. Several sperm preparation methods, swim-up (SU) and the density-gradient centrifugation (DGC) are frequently used to select for better sperm quality which also contribute to IUI failure. Sperm selection methods mainly separate the motile from the immotile sperm, eliminating the seminal plasma. The sperm motility involves the structure and function of sperm membrane in maintaining the balance of ion transport system which is regulated by the Na+, K+-ATPase, and Ca2+-ATPase enzymes. This study aims to re-evaluate the efficiency of these methods in selecting for sperm before being used for IUI and based the evaluation on sperm Na+,K+-ATPase and Ca2+-ATPase activities. Fourteen infertile men from couples who underwent IUI were involved in this study. The SU and DGC methods were used for the sperm preparation. Semen analysis was performed based on the reference value of World Health Organization (WHO) 2010. After isolating the membrane fraction of sperms, the Na+, K+-ATPase activity was defined as the difference in the released inorganic phosphate (Pi) with and without the existence of 10 mM ouabain in the reaction, while the Ca2+-ATPase was determined as the difference in Pi contents with and without the existence of 55 µm CaCl2. The prepared sperm demonstrated a higher percentage of motile sperm compared to sperm from the whole semen. Additionally, the percentage of motile sperm of post-DGC showed higher result than the sperm from post-SU. The velocity of sperm showed similar pattern with the percentage of motile sperm, in which the velocity of prepared sperm was higher than the sperm from whole semen. Furthermore, the sperm velocity of post-DGC was higher compared to the sperm from post-SU. The Na+, K+-ATPase activity of prepared sperm was higher compared to whole semen, whereas Na+, K+-ATPase activity in the post DGC was higher than post SU. The Ca2+-ATPase activity of prepared sperm was higher compared to whole semen, whereas Ca2+-ATPase activity in the post DGC was higher than post SU. The SU and the DGC methods were able to perform sperm selection by showing a high result of Na+, K+-ATPase and Ca2+-ATPase activities, moreover DGC method selected the sperm with high activities of both the Na+, K+-ATPase and Ca2+-ATPase better compared to SU method.As one of the treatment for infertility, the success rate of Intrauterine Insemination (IUI) is still relatively low. Several sperm preparation methods, swim-up (SU) and the density-gradient centrifugation (DGC) are frequently used to select for better sperm quality which also contribute to IUI failure. Sperm selection methods mainly separate the motile from the immotile sperm, eliminating the seminal plasma. The sperm motility involves the structure and function of sperm membrane in maintaining the balance of ion transport system which is regulated by the Na+, K+-ATPase, and Ca2+-ATPase enzymes. This study aims to re-evaluate the efficiency of these methods in selecting for sperm before being used for IUI and based the evaluation on sperm Na+,K+-ATPase and Ca2+-ATPase activities. Fourteen infertile men from couples who underwent IUI were involved in this study. The SU and DGC methods were used for the sperm preparation. Semen analysis was performed based on the reference value of World Health Organizatio...

Sperm Na+ K+-ATPase and Ca2+-ATPase Activities: A Potential Predictive Parameter of Sperm Motility Disorder in Infertile Men

Current highlight on the etiology of male infertility is disorder of sperm motility in which caused by ion homeostasis imbalance involving the ubiquitous multifunctional transmembrane protein, Na+K+-ATPase and Ca2+-ATPase enzymes. The emphasis of this review is evaluating the sperm Na+K+-ATPase and Ca2+ATPase activity as predictive parameters of sperm motility disorder. To this purpose, a computerized search of PubMed database was performed and obtained data were reviewed in this paper. The retrieved studies were laboratory experiments involving human and mice sperm as the subjects. Na+ K+-ATPase and Ca2+-ATPase play an essential role in sperm motility by controlling ion homeostasis. Na+ K+-ATPase maintains the intracellular pH by transporting 3 Na+ out and 2 K+ into the cell, whereas Ca2+-ATPase extrudes Ca2+ from the cell. The impairment of these enzymes and its isoforms, Na+ K+-ATPase a4 and PMCA4 expression were proved to decrease sperm motility.

Azoospermia: suatu Tinjauan Genomik

Azoospermia or the absence of sperm in semen is one of the sperm disorders that results in male infertility. There are two types of azoospermia, the first one isazoospermia caused by obstruction of the vas deferens (obstructive azoospermia) and the second one is azoospermia due to the damage of testes (nonobstructive azoospermia). The etiology of azoospermia could be genetic or non-genetic. Genetic factors may occur in genomics starting from chromosome until gene level or single nucleotide polymorphism (SNPs). At the chromosome level, there is Klinefelter’s syndrome (47, XXY) to the Y chromosome microdeletion, whereas at the gene level there is mutation of jsd, Bmp8b and other genes. At the level of SNPs, Genome Wide SNP Association Study (GWAS) had uncovered 20 SNPs which were related significantly to azoospermia. Extensive knowledge of genomics review on male infertility, is expected to promote the development of investigation and management of azoospermia.

Multi-sperm tracking using Hungarian Kalman Filter on low frame rate video

One factor of human sperm health is sperm motility. Motility is the ability of sperm to move. Sperm with healthy motility move forward promptly, not inactive and not moving in circles. In this paper, we would like to analyse sperm motility by considering the problem of multi object tracking in video sequences of human sperms. The challenges in multi-sperm tracking are many human sperms have fast and unpredictable movement In addition, the sperm have similar size and shape comparing by each others. To solve this problem, we used sperm detection in each video sequence to get the position of sperms. In the same time, the estimated sperm position is calculated based on previous tracking by using Kalman Filter. Finally the positions of detected sperms are compared to estimation results by using Hungarian assignment method. In this way, the trajectory of each sperm can be conclude. This paper analyze sperm motility qualitatively based on the resulted sperm trajectories. The experiment results were conducted on both open video data and our own low-frame-rate video data. The experiment results shows that the proposed method can handle the challenges in multi sperm tracking, create their trajectory and then analyze their behaviors.