Infertility affects approximately 15% of couples trying to conceive, and a male factor contributes to roughly half of these cases. Oxidative stress (OS) has been identified as one of the many mediators of male infertility by causing sperm dysfunction. OS is a state related to increased cellular damage triggered by oxygen and oxygen-derived free radicals known as reactive oxygen species (ROS). During this process, augmented production of ROS overwhelms the body's antioxidant defenses. While small amounts of ROS are required for normal sperm functioning, disproportionate levels can negatively impact the quality of spermatozoa and impair their overall fertilizing capacity. OS has been identified as an area of great attention because ROS and their metabolites can attack DNA, lipids, and proteins; alter enzymatic systems; produce irreparable alterations; cause cell death; and ultimately, lead to a decline in the semen parameters associated with male infertility. This review highlights the mechanisms of ROS production, the physiological and pathophysiological roles of ROS in relation to the male reproductive system, and recent advances in diagnostic methods; it also explores the benefits of using antioxidants in a clinical setting.

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Effect of oxidative stress on male reproduction.

Human embryo implantation is a three-stage process (apposition, adhesion and invasion) involving synchronized crosstalk between a receptive endometrium and a functional blastocyst. This ovarian steroid-dependent phenomenon can only take place during the window of implantation, a self-limited period of endometrial receptivity spanning between days 20 and 24 of the menstrual cycle. Implantation involves a complex sequence of signalling events, consisting in the acquisition of adhesion ligands together with the loss of inhibitory components, which are crucial to the establishment of pregnancy. Histological evaluation, now considered to add little clinically significant information, should be replaced by functional assessment of endometrial receptivity. A large number of molecular mediators have been identified to date, including adhesion molecules, cytokines, growth factors, lipids and others. Thus, endometrial biopsy samples can be used to identify molecules associated with uterine receptivity to obtain a better insight into human implantation. In addition, development of functional in vitro systems to study embryo-uterine interactions will lead to better definition of the interactions existing between the molecules involved in this process. The purpose of this review was not only to describe the different players of the implantation process but also to try to portray the relationship between these factors and their timing in the process of uterine receptivity.

Endometrial receptivity markers, the journey to successful embryo implantation.

BACKGROUND: Sperm hyperactivation is critical to fertilization, because it is required for penetration of the zona pellucida. Hyperactivation may also facilitate release of sperm from the oviductal storage reservoir and may propel sperm through mucus in the oviductal lumen and the matrix of the cumulus oophorus. Hyperactivation is characterized by high amplitude, asymmetrical flagellar bending. METHODS: This is a review of the original literature on the mechanisms that regulate hyperactivation, including physiological factors and signaling pathways. RESULTS: Computer-assisted semen analysis systems can be used to identify hyperactivated sperm by setting minimum thresholds for curvilinear velocity (VSL) and lateral head movement and a maximum threshold for path linearity. Hyperactivation is triggered by a rise in flagellar Ca(2+) resulting from influx primarily through plasma membrane CatSper channels and possibly also by release of Ca(2+) from a store in the redundant nuclear envelope. It requires increased pH and ATP production. The physiological signals that trigger the rise in Ca(2+) remain elusive, but there is evidence that the increased Ca(2+) acts through a calmodulin/calmodulin kinase pathway. Hyperactivation is considered part of the capacitation process; however, the regulatory pathway that triggers hyperactivation can operate independently from that which prepares sperm to undergo the acrosome reaction. Hyperactivation may be modulated by chemotactic signals to turn sperm toward the oocyte. CONCLUSIONS: Little is known about exactly what triggers hyperactivation in human sperm. This information could enable clinicians to develop reliable fertility assays to assess normal hyperactivation in human sperm samples.

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Control of hyperactivation in sperm

Ovarian stimulation for IVF is known to affect luteal phase function. The endometrium in IVF cycles is thus subject to an altered endocrinological environment and to a possible direct effect of the ovarian stimulation therapy. Factors influencing the endometrial receptivity in such cycles are poorly understood. Studies comparing the endometrium in IVF cycles with natural cycles as controls have shown premature secretory changes in the post-ovulatory and early luteal phase of IVF cycles, followed by a large proportion of dyssynchronous glandular and stromal differentiation in the mid-luteal phase. These findings suggest a profound modification of luteal endometrial development in stimulated cycles. This hypothesis is further supported by the demonstration of a modified endometrial steroid receptor regulation and a profound antiproliferative effect in IVF cycles. The time of maximal endometrial receptivity is defined as the implantation window and is characterized by the expression of various endometrial products, among which pinopodes, integrins and leukaemia inhibitory factor are best described. Premature expression of pinopodes and integrins are in line with the observation of precocious luteal transformation following ovarian stimulation, although the clinical relevance with respect to the establishment of a clinical pregnancy awaits further validation. Studies exploring the endometrium within the cycle of embryo transfer have shown a deleterious effect of severe peri-ovulatory maturation advancement exceeding 3 days, as no clinical pregnancies were obtained in this condition. Further unravelling of molecules involved in the implantation mechanism is needed for a better comprehension of the link between altered endometrial development and receptivity in IVF cycles.

The endometrium in stimulated cycles for IVF

A proper dialogue between spermatozoa and the egg is essential for conception of a new individual in sexually reproducing animals. Ca(2+) is crucial in orchestrating this unique event leading to a new life. No wonder that nature has devised different Ca(2+)-permeable channels and located them at distinct sites in spermatozoa so that they can help fertilize the egg. New tools to study sperm ionic currents, and image intracellular Ca(2+) with better spatial and temporal resolution even in swimming spermatozoa, are revealing how sperm ion channels participate in fertilization. This review critically examines the involvement of Ca(2+) channels in multiple signaling processes needed for spermatozoa to mature, travel towards the egg, and fertilize it. Remarkably, these tiny specialized cells can express exclusive channels like CatSper for Ca(2+) and SLO3 for K(+), which are attractive targets for contraception and for the discovery of novel signaling complexes. Learning more about fertilization is a matter of capital importance; societies face growing pressure to counteract rising male infertility rates, provide safe male gamete-based contraceptives, and preserve biodiversity through improved captive breeding and assisted conception initiatives.

https://www.physiology.org/doi/pdf/10.1152/physrev.00028.2010

Calcium Channels in the Development, Maturation, and Function of Spermatozoa

https://www.fertstert.org/article/S0015-0282(99)00605-6/pdf

Endometrial dating and determination of the window of implantation in healthy fertile women.

Serum inhibin B may be a reliable marker of the presence of testicular spermatozoa in patients with nonobstructive azoospermia

Summary. Prolonged incubation of human spermatozoa can have deleterious effects on sperm function. The aim of this paper was to describe the effects of a prolonged in vitro incubation, under similar conditions to those employed in human assisted reproduction, on various sperm functional parameters, and to investigate the effect of an antioxidant (catalase) on this system. Freshly collected ejaculates from 20 healthy donors were studied. Samples were divided into two aliquots: the first was incubated with Ham's F10 containing 3.5% HAS, and the second was incubated in the same medium plus catalase (100 units ml−1). All experiments were carried out with spermatozoa isolated using the swim-up technique. Spermatozoa recovered from the supernatant after 1 h (T1) of incubation in 5% CO2 in air at 37 °C, and after 5 h (T6), 23 h (T24) and 47 h (T48), were evaluated for concentration, motion parameters including hyperactivation (computer-assisted analysis), viability, ATP concentration, reactive oxygen species (ROS) generation, DNA integrity (acridine orange), and acrosome reaction (AR). The major alteration observed in sperm function during the prolonged in vitro incubation was a reduction in the number of motile spermatozoa, together with an impairment in the quality of sperm movement. ROS levels increased with the incubation time. No substantial modifications of sperm viability, chromatin condensation and AR inducibility were observed. The addition of catalase to the medium, while keeping ROS values within baseline levels, did not prevent the loss of motility or the corresponding increase in ATP.

Effects of long‐term in vitro incubation of human spermatozoa: functional parameters and catalase effect

BACKGROUND: Human semen is composed of a heterogeneous population of sperm with varying degrees of structural and functional differentiation and normality, which result in subpopulations of different quality. METHODS: Using a discontinuous Percoll gradient, we separated three subsets of sperm [(45%; L45), (65%; L65) and (90%; L90) fractions] from normozoospermic human semen samples from healthy donors and proceeded to characterize their morphology, motility and hyperactivation, as well as their ability to undergo tyrosine phosphorylation under capacitating conditions. RESULTS: As expected, sperm isolated from the lowest density layer (L45) showed the poorest quality, displaying the smallest percentage of morphologically normal and motile sperm. During a capacitating incubation, this subset of cells also showed deficient capacity to undergo hyperactivation and protein tyrosine phosphorylation. Conversely, sperm isolated from the other layers (L65 and L90) showed a time-dependent progressive increment in tyrosine phosphorylation, establishing statistically significant differences with sperm from L45. The tyrosine phosphorylation deficiency of L45 sperm could be overcome when sperm from that fraction were stimulated with activators of the cAMP-dependent kinase (PKA) pathway (dbcAMP + pentoxifylline), pointing to the sperm's plasma membrane as the main site of such deficiency. CONCLUSIONS: Poor quality sperm isolated from a Percoll gradient display an intrinsic tyrosine phosphorylation deficiency, possibly caused by a plasma membrane defect, which is associated with their inability to undergo normal capacitation and, ultimately, acquire optimal fertilizing potential.

Human sperm subpopulations: relationship between functional quality and protein tyrosine phosphorylation

Poor-quality sperm show reduced capacity to undergo capacitation-induced protein tyrosine phosphorylation and hyperactivation. Given that these deficiencies can be overcome by membrane-permeant stimulators of the cAMP-dependent kinase system, we hypothesize that the main defect underlying these deficiencies resides on the sperm plasma membrane. Spermatozoa from semen samples obtained from 15 consenting healthy donors were separated in 2 subpopulations, L45 (first interface) and L90 (pellet), using a 45:65:90 ISolate gradient centrifugation method. These sperm fractions were studied before and after a 6-hour capacitating incubation for sperm motion parameters (computer-assisted analysis), including hyperactivation, protein tyrosine phosphorylation (immunofluorescence), membrane fluidity (Laurdan fluorescence), and sterol and phospholipid content (high-performance thin-layer chromatography). In summary, data indicate that L45 (poor-motility) spermatozoa present an excess of cholesterol and desmosterol, which impairs the normal increase in membrane fluidity during capacitation and its consequent activation of protein tyrosine phosphorylation and hypermotility. Therefore, a defect in membrane composition and dynamics is underlying human sperm biochemical and functional deficiencies related to inadequate capacitation.

High Cholesterol Content and Decreased Membrane Fluidity in Human Spermatozoa Are Associated With Protein Tyrosine Phosphorylation and Functional Deficiencies

J.C. Calamera

Papers

Endometrial dating and determination of the window of implantation in healthy fertile women.

Serum inhibin B may be a reliable marker of the presence of testicular spermatozoa in patients with nonobstructive azoospermia

Effects of long‐term in vitro incubation of human spermatozoa: functional parameters and catalase effect

Human sperm subpopulations: relationship between functional quality and protein tyrosine phosphorylation

High Cholesterol Content and Decreased Membrane Fluidity in Human Spermatozoa Are Associated With Protein Tyrosine Phosphorylation and Functional Deficiencies