Gene Expression Changes in the Ovary Mediate Non-Anovulatory Mechanisms of Contraception with Levonorgestrel.

BACKGROUND: Emergency contraception with levonorgestrel (LNG) is a viable option to prevent unintended pregnancies. Although the efficacy of LNG as an anovulatory agent decreases as treatment approaches ovulation, it still provides some contraceptive benefits. AIM: To better understand the contraceptive mechanisms of LNG in ovulatory subjects. METHODS: We conducted a study on Wistar rats that received a single dose of LNG (0.01 or 0.05 mg/kg) on the morning of proestrus before ovulation and evaluated its effects on ovarian gene expression, ovulation, and implantation. RESULTS: Our findings showed changes in the expression of genes involved in follicular development and oocyte quality. Pregnancy rates - as an indicator of ovulation - and embryo implantation were significantly lower than those in the control group. CONCLUSIONS: This study suggests that LNG alters regulatory factors in the ovary that are essential for the development of competent fertilizable oocytes, highlighting the non-anovulatory mechanisms by which levonorgestrel may regulate fertility and suggesting that it could be a novel observation that contributes to the understanding of emergency contraception in humans.

Sperm mRNAs as potential markers of male fertility.

Advances in transcriptomic technologies are contributing to an increased understanding of the role of spermatozoal RNA in sperm physiology. Although sperm transcriptomic studies have delivered large amounts of valuable information, no new male fertility biomarkers have emerged from such studies to date. This review summarizes current knowledge about the potential relevance of certain mRNA as biomarkers, focusing on comparative studies of human spermatozoa transcriptomic profiles from fertile and pathological semen samples. Asthenozoospermia is the semen aberrant condition that has been most exhaustively investigated to date. We cross-analyzed findings from three different studies on the transcriptome of asthenozoospermic semen samples and identified 100 transcripts that were consistently differentially expressed and that consequently are candidates for characterizing the molecular source of this sperm anomaly. The potential use of sperm mRNAs as predictors of outcomes of assisted reproductive technologies (ART) is also reviewed. Improving the understanding of the human spermatozoa mRNA content is expected to improve the evaluation and diagnosis of infertile men, and ultimately facilitate the selection of the best treatment to overcome infertility.

Effects of Semen Processing on Sperm Function: Differences between Swim-Up and Density Gradient Centrifugation.

PURPOSE: Andrology research has evolved notoriously in the latest years, particularly since male factor contribution to couple infertility has been undoubtedly demonstrated. However, sperm function investigations results are sometimes contradictory, probably as a result of the use of different sperm processing techniques. In this work, we underwent a systematic functional comparison of human sperm samples simultaneously processed by swim-up and density gradient centrifugation, which are the preferred sperm processing methods used in basic and clinical laboratories. MATERIALS AND METHODS: To compare functional characteristics of sperm isolated by swim-up and density gradient centrifugation followed by incubation at different times under capacitating conditions. RESULTS: Semen samples processed in parallel by these two procedures resulted in sperm preparations with significant differences in redox state, spontaneous intracellular calcium oscillations, hyperactivation, protein tyrosine phosphorylation, and acrosome reaction responsivity to calcium ionophore. Such differences showed time-dependent specific patterns for spontaneous intracellular calcium oscillations, hyperactivation and protein tyrosine phosphorylation. Sperm retrieved by density gradient centrifugation showed more hyperactivation and tyrosine phosphorylation than swim-up sperm, suggesting a higher degree of capacitation. CONCLUSIONS: Our results account for functional differences observed in spermatozoa processed with these two methods and therefore may contribute to a better interpretation of outcomes obtained in different laboratories as well as to improve experimental designs aimed to study sperm physiology and fertility potential.

Proteomic characterization of human sperm plasma membrane-associated proteins and their role in capacitation.

BACKGROUND: Plasma membranes of ejaculated sperm are covered by epididymal and accessory glands secreted proteins that must be released from sperm surface during the female reproductive tract passage in order to capacitate and fertilize the oocyte. OBJECTIVES: As human sperm plasma membrane-associated proteins (SMAP) have not yet been investigated, the aim of this study was to characterize the SMAP released during in vitro human capacitation and to study their possible role as decapacitation factors. MATERIALS AND METHODS: SMAP were characterized by 2-dimensional electrophoresis and mass spectrometry analysis. Besides, we explored SMAP effects on motility, protein tyrosine phosphorylation, and calcium ionophore-induced acrosome reaction of spermatozoa either incubated for 6 h in capacitating medium ± SMAP or for 5 h in capacitating medium alone followed by incubation for 1 h ± SMAP. RESULTS: Mass spectrometry analysis allowed the identification of 29 proteins, all of which have previously been identified in the human seminal fluid. Spermatozoa incubated for 6 h under capacitating conditions in the presence of the SMAP showed a significant decrease in the incidence of non-progressive motility, hyperactivation, protein tyrosine phosphorylation, and calcium ionophore-induced acrosome reaction. However, spermatozoa incubated for 5 h in capacitating medium and further incubated for 1 h with the SMAP showed a lower percentage of spermatozoa with non-progressive motility and hyperactivated cells but no effects on protein tyrosine phosphorylation were detected. DISCUSSION AND CONCLUSIONS: Our results indicate that SMAP inhibit the progress of human sperm capacitation, but only motility changes related to capacitation may be reversed by these proteins. The study of the identified proteins on sperm function and their mechanisms of action on this cell may contribute to the understanding of their role during capacitation.

Proteins from male and female reproductive tracts involved in sperm function regulation.

SummarySpermatogenesis is a dynamic process that culminates in the production of mature spermatozoa in the seminiferous tubules of sexually mature animals. Although sperm leaving the testis are fully differentiated, they must further undergo two additional maturation steps before acquiring the capability to fertilize the egg. Such processes take place during the epididymal residency and transport in the seminal fluid during ejaculation and, after delivery into the female reproductive tract, during the journey aiming the encountering the egg in the oviduct. Throughout this trip, spermatozoa are exposed to different reproductive fluids whose molecular compositions regulate the progress towards obtaining a fertilized competent cell. This review summarizes the evidence obtained so far supporting the participation of male and female reproductive tract-derived proteins in the modulation of sperm fertilizing ability and discusses the mechanisms by which such regulation may be accomplished.

Proteomic changes in uterine flushings after levonorgestrel treatment and effects on sperm function.

When levonorgestrel (LNG) is given for emergency contraception during the follicular phase, it not only inhibits or delays ovulation, but also induces changes in endometrial secretions that modulate sperm functionality. In order to characterize the female reproductive tract secreted molecules that may affect human spermatozoa, we analyzed changes in the protein content of uterine flushings obtained from women during the periovulatory phase of a control and a LNG-treated menstrual cycle. Lectin affinity analysis and 2D gel electrophoresis of uterine samples showed changes in protein glycosylation patterns and the presence of 31 differentially expressed proteins (8 upregulated and 23 downregulated). Mass spectrometry and Western blot analyses of the differential expressed proteins showed lactotransferrin (LTF) as one of the upregulated molecules by LNG. In this study, LTF exhibited significant dose-related effects on sperm functionality, particularly a decrease of calcium ionophore-induced acrosome reaction and protein tyrosine phosphorylation. Overall, the results indicated that LNG promoted changes in the proteome of uterine secretions that might compromise human sperm capacitation. These data further support the participation of other mechanisms of action of LNG as emergency contraceptive, in addition to those on ovulation.

Uterine flushings from women treated with levonorgestrel affect sperm functionality in vitro.

Levonorgestrel (LNG), a synthetic 19 nor-testosterone derivative, is widely used for emergency contraception. It is well known that LNG prevents ovulation only when given prior to the surge of serum luteinizing hormone (LH) during the periovulatory phase of the menstrual cycle. This observation suggests that LNG, given its contraceptive efficacy, has additional effects other than those affecting ovulation. In this study, we have evaluated the effects on human sperm functionality of uterine flushings (UF) obtained from women at day LH + 1 of a control cycle (CTR-LH + 1) and after receiving LNG (LNG-LH + 1) two days before the surge of LH. Human sperm from normozoospermic donors were incubated with UF and protein tyrosine phosphorylation, sperm motility, acrosome reaction as well as zona pellucida (ZP) binding capacity were assessed. A significant decrease in total motility and tyrosine phosphorylation accompanied by an increase on spontaneous acrosome reaction was observed when sperm were incubated in the presence of LNG-LH + 1. None of these effects were mimicked by purified glycodelin A (GdA). Moreover, the addition of UF obtained during the periovulatory phase from LNG-treated women or the presence of purified GdA significantly decreased sperm-ZP binding. The data were compatible with changes affecting sperm capacitation, motility and interaction with the ZP. These results may offer evidence on additional mechanisms of action of LNG as an emergency contraceptive.