2018 Mar 15. doi: 10.1007/s10103-018-2476-y. [Epub ahead of print]

Has the time come to include low-level laser photobiomodulation as an adjuvant therapy in the treatment of impaired endometrial receptivity?

El Faham DA¹, Elnoury MAH², Morsy MI², El Shaer MA³, Nour Eldin GM⁴, Azmy OM⁵.

Author information

1: Departement of Obstetrics and Gynecology, National Research Centre, Cairo, Egypt. drdody_as@hotmail.com.
2: Departement of Medical Applications, National Institute of Laser Enhanced Science, Cairo University, Cairo, Egypt.
3: Departement of Pathology, National Research Centre, Cairo, Egypt.
4: Departement of Cytogenetics, National Research Centre, Cairo, Egypt.
5: Departement of Obstetrics and Gynecology, National Research Centre, Cairo, Egypt.

Abstract

Low-level laser therapy (LLLT) has been used in photobiomodulation to promote healing, regenerate, and restore tissue function. Women with persistent thin endometrium were assumed to encounter diminished activity in the regenerative and functional capacity of their endometrium. The aim of this study was to examine the ability of LLLT in 635 nm wavelength to enhance the proliferation and gene expression of in vitro cultured endometrial cells. Single (SE) versus multiple exposures (ME) to LLLT were examined in the study groups and compared to controls. A fluence dose of 4.27 J/cm² for 16 min was given once in the SE or divided in three equal sessions in days 2, 4, and 6 of the culture in ME. Cellular response was evaluated by measuring viable cell numbers and surface area. Pattern of MUC1, ITGA5, ITGB3, LIF, and PTEN gene expression was assessed using the qRT-PCR. Greater numbers of cells were found in both study groups (P < 0.001) as compared to controls. The surface area of cells at the end of culture phase was highly significant (P < 0.001) in ME when compared to SE and controls. A statistically significant difference was found in terms of gene expression in both irradiated groups (P < 0.001) as compared to controls, although greater difference in PTEN tumour suppressor gene (Phosphatase and tensin homolog) expression was toward ME. The introduction of LLLT to the armamentarium of infertility will serve as a new adjuvant therapy in this field. The current study proofed that LLLT was able to increase the proliferative and functional capacity of cultured endometrial cells.

KEYWORDS:

Cell culture; Endometrial receptivity; Gene expression; Infertility; Low-level laser therapy; Thin endometrium

2017 Mar 1;20(2):307-312. doi: 10.1515/pjvs-2017-0037.

Improvement of dairy cow embryo yield with low level laser irradiation.

Palubinskas G, Žilaitis V¹, Antanaitis R¹.

Author information

1

Abstract

The goal of this study is to estimate the effects of low-level laser irradiation (LLLI) on the superovulatory response according to the number of corpora lutea (CL), follicles (F) and the embryo yield. In recent years, while searching for new, more efficient and organic methods to improve superovulatory response and embryo yield with respect to the conventional methods, low-level laser irradiation (LLLI) is a more sensitive and less costly technology that can be used to improve animal reproduction, namely, artificial insemination and the embryo production system. The dairy-cow donors were treated for superovulation with Pluset®, at any time during the oestrus cycle, and the total dose per donor was 700 IU. The first group of the donors (n=25), test group (TG), was irradiated on the sacroiliac area for 180 seconds per day, from the 1st to 11th superovulatory treatment (ST) days in a row, with LLLI in the 870-970-nm wavelength, 65.93 J/cm dose, frequencies in the 20-2000 Hz range and pulse durations commonly in the range of about 1 second. For the second control group (CG) (n=25), the ST was performed without LLLI. After the ST, The mean number of CL in the right side ovaries in the TG was 25.43% (p<0.05) greater than in those of the CG. The number of total recovered and transferable embryos was greater in the TG compared with the CG by 28.97% (p<0.05) and 15.8% (p>0.05), respectively. With respect to conventional methods, LLLI can be used to improve the superovulatory response and embryo yield as a supplementary environment and animal-friendly method of treatment.

2016 Aug;31(6):1245-50. doi: 10.1007/s10103-016-1966-z. Epub 2016 Jun 7.

Effects of photobiomodulation therapy (PBMT) on bovine sperm function.

Siqueira AF¹, Maria FS¹, Mendes CM^1,², Hamilton TR¹, Dalmazzo A³, Dreyer TR⁴, da Silva HM⁴, Nichi M³, Milazzotto MP⁴, Visintin JA², Assumpção ME⁵.

Author information

1: Laboratory of Spermatozoa Biology, School of Veterinary Medicine and Animal Science, Department of Animal Reproduction, University of São Paulo, São Paulo, Brazil.
2: Laboratory of in vitro Fertilization, Cloning and Animal Transgenesis, School of Veterinary Medicine and Animal Science, Department of Animal Reproduction, University of São Paulo, São Paulo, Brazil.
3: Laboratory of Andrology, School of Veterinary Medicine and Animal Science, Department of Animal Reproduction, University of São Paulo, São Paulo, Brazil.
4: Centro de Ciências Naturais e Humanas (CCNH), Federal University of ABC, Santo André, Brazil.
5: Laboratory of Spermatozoa Biology, School of Veterinary Medicine and Animal Science, Department of Animal Reproduction, University of São Paulo, São Paulo, Brazil. meoaa@usp.br.

Abstract

Fertilization rates and subsequent embryo development rely on sperm factors related to semen quality and viability. Photobiomodulation therapy (PBMT) is based on emission of electromagnetic waves of a laser optical system that interact with cells and tissues resulting in biological effects. This interaction is mediated by photoacceptors that absorb the electromagnetic energy. Effects are dependent of irradiation parameters, target cell type, and species. In sperm, PBMT improves several features like motility and viability, affecting sperm aerobic metabolism and energy production. The aim of this study was to investigate, under same conditions, how different output powers (5, 7.5, and 10 mW) and time of irradiation (5 and 10 min) of laser (He-Ne laser, 633 nm) may affect frozen/thawed bovine sperm functions. Results showed significant effects depending on power while using 10 min of irradiation on motility parameters and mitochondrial potential. However, no effect was observed using 5 min of irradiation, regardless of power applied. In conclusion, PBMT is effective to modulate bovine sperm function. The effectiveness is dependent on the interaction between power applied and duration of irradiation, showing that these two parameters simultaneously influence sperm function. In this context, when using the same fluency and energy with different combinations of power and time of exposure, we observed distinct effects, revealing that biological effects should be also based on simple parameters rather than only composite parameters such as fluency, irradiance and energy. Laser irradiation of frozen/thawed bovine semen led to an increase on mitochondrial function and motility parameters that could potentially improve fertilityrates.

2016 May;31(4):695-704. doi: 10.1007/s10103-016-1911-1. Epub 2016 Feb 25.

Low–level laser therapy to recovery testicular degeneration in rams: effects on seminal characteristics, scrotal temperature, plasma testosterone concentration, and testes histopathology.

Alves MB¹, de Arruda RP², Batissaco L¹, Florez-Rodriguez SA¹, de Oliveira BM¹, Torres MA³, Ravagnani GM³, Lançoni R², de Almeida TG¹, Storillo VM¹, Vellone VS¹, Franci CR⁴, Thomé HE¹, Canella CL¹, De Andrade AF³, Celeghini EC⁵.

Author information

1: Laboratory of Teaching and Research in Pathology of Reproduction, Center of Biotechnology in Animal Reproduction, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo (USP), Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, SP, Brazil.
2: Laboratory of Semen Biotechnology and Andrology, Center of Biotechnology in Animal Reproduction, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo (USP), Pirassununga, SP, Brazil.
3: Laboratory of Andrology and Embryo Technology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo (USP), Pirassununga, SP, Brazil.
4: Laboratory of Neuroendocrinology and Reproduction, Department of Physiology – Faculty of Medicine, University of Sao Paulo (USP), Ribeirao Preto, SP, Brazil.
5: Laboratory of Teaching and Research in Pathology of Reproduction, Center of Biotechnology in Animal Reproduction, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo (USP), Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, SP, Brazil. celeghin@usp.br.

Abstract

The aim of this study was to investigate the efficiency of low–level laser therapy (LLLT) to recovery testicular degeneration in rams. In the first study, rams were induced to testicular degeneration by scrotal insulation, and then, they were treated using LLLT at 28 J/cm(2) (INS28) or 56 J/cm(2) (INS56) energy densities. Sperm kinetics, morphology, and membranes integrity as well as proportion of lumen area in seminiferous tubule were assessed. In the second study, rams were submitted or not to scrotal insulation and treated or not by the best protocol of LLLT defined by experiment 1 (INS28). In this study were evaluated sperm kinetics, morphology, membranes integrity, ROS production, and DNA integrity. Testosterone serum concentration and proportion of lumen area in seminiferous tubule were also analyzed. Insulation was effective in promoting sperm injuries in both experiments. Biostimulatory effect was observed in experiment 1: INS28 presented smaller proportion of lumen area (P?=?0.0001) and less degeneration degree (P?=?0.0002). However, in experiment 2, there was no difference between the groups (P?=?0.17). In addition, LLLT did not improve sperm quality, and there was a decreasing for total and progressive motility (P?=?0.02) and integrity of sperm membranes (P?=?0.01) in LLLT-treated groups. Moreover, testosterone concentration was not improved by LLLT (P?=?0.37). Stimulation of aerobic phosphorylation by LLLT may have led to a deregulated increase in ROS leading to sperm damages. Thus, LLLT at energy of 28 J/cm(2) (808 nm of wavelength and 30 mW of power output) can induce sperm damages and increase the quantity of cells in seminiferous tubule in rams.

2015 Mar 31;87(1):14-9. doi: 10.4081/aiua.2015.1.14.

Light-emitting diode exposure enhances sperm motility in men with and without asthenospermia: preliminary results.

Salama N1, El-Sawy M.

Author information

1Department of Urology, Alexandria Faculty of Medicine, Alexandria. nadersalama58@yahoo.com.

Abstract

OBJECTIVE:

To evaluate the effect of lightemitting diode (LED) on sperm motility in men with and without asthenospermia.

MATERIAL AND METHODS:

Semen samples from 27 men were assessed and washed. An aliquot was taken from each sample as a control. The remaining amount was exposed to red LED for 2, 5 and 10 minutes. Sperm motility from the test and control tubes were re-checked at the end of each time interval. In 11 of these 27 samples, the same protocol was repeated without sperm washing. Evaluation of sperm creatine kinase (CK) activity, hypoosmotic swelling (HOS) test and aniline blue staining (ANBS) were undertaken after phototherapy in additional 15 samples.

RESULTS:

Progressive sperm motility increased significantly after LED treatment at the different time intervals whether in washed (p = 0.000) or non-washed (p = 0.003) samples. The amount of the increase in motility in washed aliquots was significantly more (p = 0.000) than in naive semen. Sperm CK activity increased, but was not significant whilst there were no changes regarding HOS and ANBS.

CONCLUSION:

Red LED is a promising safe tool to boost sperm motility in vitro. This may have a great implication on maximizing the possibilities and outcomes of intrauterine insemination trials.

. 2015; 10(3): e0121487.

Published online 2015 Mar 17. doi: 10.1371/journal.pone.0121487

PMCID: PMC4364308

The Effect of Low-Level Laser Irradiation on Sperm Motility, and Integrity of the Plasma Membrane and Acrosome in Cryopreserved Bovine Sperm

Guilherme Henrique C. Fernandes,¹ Paulo de Tarso Camillo de Carvalho,^1,^2,^* Andrey Jorge Serra,¹ André Maciel Crespilho,³ Jean Pierre Schatzman Peron,⁴ Cristiano Rossato,⁴ Ernesto Cesar Pinto Leal-Junior,^1,² and Regiane Albertini^1,²

Roberto Amendola, Academic Editor

¹Postgraduate Program in Rehabilitation Sciences, Universidade Nove de Julho (UNINOVE), São Paulo, SP, Brazil

²Postgraduate Program in Biophotonics, Universidade Nove de Julho (UNINOVE), São Paulo, SP, Brazil

³Postgraduate Program in Veterinary Medicine—Universidade de Santo Amaro (UNISA) São Paulo, São Paulo, SP, Brazil

⁴Instituto de Ciências Biomédicas da Universidade de São Paulo—USP—São Paulo, São Paulo, SP, Brazil

ENEA, ITALY

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: PTCC RA ECLJ AJS. Performed the experiments: GHCF JPSP CR AMC. Analyzed the data: AJS PTCC RA. Contributed reagents/materials/analysis tools: JPSP AMC CR. Wrote the paper: PTCC GHCF ECLJ. Edited the final format of the paper: GHCF PTCC. Discussion of the paper: PTCC GHCF. Edited the final version of the manuscript: PTCC GHCF ECLJ.

* E-mail: rb.evoninu@osrat.oluap

Author information ? Article notes ? Copyright and License information ?

Received 2014 Dec 8; Accepted 2015 Feb 2.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

This article has been cited by other articles in PMC.

Abstract

Background and Objective

Freezing changes sperm integrity remarkably. Cryopreservation involves cooling, freezing, and thawing and all these contribute to structural damage in sperm, resulting in reduced fertility potential. Low-level laser irradiation (LLLI) could increase energy supply to the cell and cause reactive oxygen species reduction (ROS), contributing to the restoration of oxygen consumption and adenosine triphosphate synthesis (ATP) in the mitochondria. Our goal was to analyze the effects of low-level laser irradiation on sperm motility and integrity of the plasma membrane and acrosome in cryopreserved bovine sperm.

Study Design/Materials and Methods

We analyzed 09 samples of bull semen (Bos taurus indicus), divided into three groups: a control group without laser irradiation, a 4J group subjected to a laser irradiation dose of 4 joules, and a 6J group subjected to dose of 6 joules. Samples were divided for the analysis of cell viability and acrosomal membrane integrity using flow cytometry; another portion was used for motion analysis. Irradiation was performed in petri dishes of 30 mm containing 3 ml of semen by an aluminum gallium indium phosphide laser diode with a wavelength of 660 nm, 30 mW power, and energy of 4 and 6 joules for 80 and 120 seconds respectively. Subsequently, the irradiated and control semen samples were subjected to cryopreservation and analyzed by flow cytometry (7AAD and FITC-PSA) using the ISAS – Integrated Semen Analysis System.

Results

Flow cytometry showed an increase in the percentage of live sperm cells and acrosome integrity in relation to control cells when subjected to irradiation of low-power laser in two different doses of 4 and 6 joules (p < 0.05). In the analysis of straightness, percentage of cell movement, and motility, a dose of 4 joules was more effective (p < 0.05).

Conclusion

We conclude that LLLI may exert beneficial effects in the preservation of live sperm. A dose of 4 joules prior to cryopreservation was more effective than a dose of 6 joules in preserving sperm motility.

Introduction

Techniques in animal reproduction, such as artificial insemination (AI), are constantly used to increase the quality and quantity of genetic and phenotypically superior calves [1], [2]. The Artificial Insemination in Fixed Time (TAI) allows cows to be inseminated without determining whether they are in heat, because the technique itself induces ovulation. The TAI is widely used to improve genetic quality and herd production volume. The efficiency of the technique is dependent on the semen quality fertilizer and pre-frozen state for post-thaw, which lead to adequate semen motility, vigor, and high viability [3], [4], [5] [6].

Sperm integrity shows remarkable changes with freezing. The cooling, freezing, and thawing involved in cryopreservation all contribute to structural damage and reduced function in sperm, resulting in reduced fertility potential [6], [7], [8].

The cryopreservation process induces morphological changes in semen and damages the plasma membrane, acrosome, and mitochondria. These changes are sufficient to adversely affect the fertilizing capacity of the semen and they significantly accentuate the production of adenosine triphosphate (ATP) and lead to cell death [9]. The sperm plasma membrane regulates the intracellular calcium concentration, particularly the calcium pump by ATP-dependent sodium/calcium and the voltage-dependent calcium channel. [10]. Intracellular calcium movements play a vital role in cell proliferation and in mammalian spermatozoa have a pivotal role in control of sperm motility and acrosome reaction. [11]

Cryopreservation of bull semen adversely affects the sperm membrane integrity, thereby reducing the ability to fertilize the processed sample [12]. In this perspective, effective techniques are needed to protect sperm from adverse effects of cryopreservation.

The low- power laser irradiation of spermatozoa can increase sperm motility as well as velocity can be improved by He-Ne laser irradiation. The first published studies dating back to the year 1984. [13] According to Huang et al. [14], the first Law of Photochemistry says that light photons are absorbed by photoreceptors or chromophores. The low-level laser mechanism at the cellular level has been attributed to the absorption of monochromatic visible radiation and near infrared (NIR) radiation by the cell respiratory chain components. The low-level laser has become an alternative to modulate various biological processes. Depending on the wavelength, dosage, and condition of the irradiated tissue, the laser can induce an anti-inflammatory effect, reducing pain, and accelerating cell proliferation [15].

The biological mechanisms of interaction of the low-power laser aren’t totally known, however, it is known that different kinds of cells don’t behave at same way when irradiated by the same wavelength. For this reason, it is difficult to extrapolate the effects from one cell type to another, but it can be said that at the molecular level, the activation of certain receptors and messengers determine universal biological responses [16].

Therefore, the present study aimed to investigate the effects of low-level laser irradiation on sperm motility, and integrity of the plasma membrane and acrosome in cryopreserved bovine sperm.

Methods and Materials

All semen handling procedures were performed in accordance with standards established by the Brazilian College of Animal Reproduction (CBRA). The experimental procedures were approved by the Research Ethics Committee of the Universidade Nove de Julho- UNINOVE n.0047/2014 and are in accordance with current legislation.

Animals

We used 09 semen samples from Nelore bulls (Bos taurus indicus), with ages ranging from 24 to 50 months, from the Central Artificial Insemination TAIRANA, (Rod Raposo Tavares, 563 KM.—Presidente Prudente, SP—Brazil). The animals were fed all year round with mineral supplementation and balanced feed and water ad libitum.

Collection and processing of semen samples

The semen collection was performed according to the health and safety criteria established by the Brazilian College of Animal Reproduction (CBRA). After collection, the semen was taken immediately to the laboratory for sample preparation and physical and morphological analysis. Semen samples were stored in formalin-saline for evaluation of concentration and sperm morphology. The samples of semen used were those at the 50^th percentile and above in motility, sperm concentration greater than 200 × 10⁶/mL, and ejaculate volume greater than 5.0 ml.

Evaluation of sperm concentration

Sperm concentration was determined using Neubarhemocytometer. Samples of semen were diluted with BotuBOV (Botupharma, Botucatu SP—Brazil), until obtaining a final concentration 25 × 10⁶ sperm / mL. The number of spermatozoa was counted in 10 squares with the help of manual counter grid was located with 200X magnification under a phase contrast microscope.

Experimental design

Viable semen samples obtained from 9 animals were divided into 3 groups: a control group without LLLI (n = 18 Straws); a 4J group treated with LLLI with 4 joules (n = 18 Straws); and a 6J group, exposed to LLLI with 6 joules (n = 18 Straws). The resulting samples were divided into 27 samples to examine cell viability and acrosomal membrane integrity and 27 samples for motion analysis.

Laser irradiation

The laser used was the Aluminum gallium indium phosphide (AlGaInP) DMC brand, model Photon Laser III (DMC, São Carlos, SP—Brazil) with a wavelength of 660 nm, and variable power from 30 to 100 mW. For this study the equipment was programmed for a power of 30 mW, beam area 0.028 cm and 4 joules of energy for the 4J group (133 seconds of irradiation), and 6 joules for the 6J group (200 seconds of irradiation). Since we performed irradiations directly to petri dishes, we decided to use lower power output of laser device we had in laboratory. The irradiation procedure was carried out in petri dishes of with a total area of 23.6 cm², containing 3 mL of semen with the laser probe was positioned perpendicularly to the plate, within 1 cm, so that the laser beam would illuminate the entire area of the plate without spreading to the outside area, resulting in a power density of 0.0012 W / cm². We choose to perform the irradiation before freezing of samples in order to protect samples of freezing procedures. In order to ensure a uniform procedure was made a protective Ethylene Vinyl Acetate (EVA) black, involving the entire outer area of the plate. The measurement of delivered energy was performed using the Newport multifunction optical meter (Model 1835C, Newport Corporation, Irvine, CA, USA). The samples of control group were not exposed to irradiation, but were exposed to same experimental conditions of all other groups, including the time before freezing procedure start to be performed.

Cryopreservation

The samples were packaged in French 0.5-mL straws (medium), previously identified with the animal number, the group to which it belongs and collection date. Filling and sealing of the straws was done by an automated system. The semen was cryopreserved (196°C negative) using the Digitcool IMV (IMV—L’Aigle, France). The straws were removed from the machine and immersed in liquid nitrogen, placed in identified according raquis with the experimental group and stored in cryogenic cylinders.

Analysis of sperm acrosomal integrity and living cells by flow cytometry

The samples were thawed in a water bath at 37°C for 30 seconds and semen placed in a microfuge preheated to 37°C. A 0.5-mL aliquot was removed from each treatment and added to 1.5 mL of 1× PBS solution. An aliquot of 300 ?L was withdrawn from the first solution and added to 1.0 mL then centrifuged at 300 g for 10 minutes (Model Mini Spin Minicentrífuga Plus, Eppendorf) in microtubes. The supernatant was discarded and the pellet resuspended in 240 ?L based on the second solution and 80 ?L resuspended in PBS. Thus, the samples showed a concentration of 25 × 10⁶ sperm/mL. Then 2 ?L of 7-amino actinomycin (7AAD) was added, associated with 2 ?L of fluorescein isothiocyanate (FITC) + Pisium Sativum Agglutinin (PSA) in the 80 ?L sample, and incubated for 8 minutes at room temperature and protected from light. [17], [18]

The analysis of acrosome integrity and live sperm cells were analyzed by flow cytometry that was performed using an Accuri C6 flow cytometer (Accuri Cytometers, Inc. Ann Arbor, MI USA), equipped with a blue and a red laser, two scatter detectors, and four fluorescence detectors (FL1 533/30 nm; FL2 585/40 nm; FL3>670 nm and FL4 675/25 nm) whose range displayed data across 6.2 logs. For this we use fluorescein isothiocyanate (FITC) + Pisium Sativum Agglutinin (PSA) fluorescence was detected at 515–545 nm Fluorescence detector 1 (Fl 1) and 7-amino actinomycin (7AAD) fluorescence was detected at 640 and 680 nm Fluorescence detector 3 (Fl 3). The data analyzed using the Accuri software (CFlow Plus, Ver. 1.0.202.1). The forward scatter and side scatter were plotted, as well as florescence detected by plotting detection on FL-1 versus FL-3. Gating and fluorescence compensation values were set after data collection. A total of 10000 events were analyzed for each sample.

Sperm motility analysis

Sperm motility was evaluated with the ISAS—Integrated Semen Analysis System. The samples were thawed in a water bath at 37°C for 30 seconds, and 2 ?L of the sample were placed in the previously heated reading chamber. Images were captured by a camera attached to a microscope connected to a computer and then analyzed in real time by the software. For this, each sperm cell was identified and its trajectory reconstructed. Parameters analyzed were fast cells; total motility (MT-%); progressive motility (MPRO-%); path velocity (VAP—microns / s) defined by the total distance of the path of each cell divided by the time elapsed; progressive velocity (VSL—microns / s), which is the distance traveled between the beginning and end of the path divided by the elapsed time; curvilinear velocity (VCL—microns / s); the lateral displacement of the head (ALH—microns), the average width of the head and the oscillation of its movement; beat frequency (BCF—Hz), the frequency at which the sperm crosses a path in each direction; straightness (STR-%), measuring the straight path of the sperm cell, is the ratio of VSL / VAP; and linearity (LIN-%), corresponding to the direction of travel, is the ratio of VSL / VCL. [19], [20]

Statistical Analysis

The Kolmogorov specification test was used to verify the normal statistical distributions and all data were expressed with means ± standard deviation. One-way ANOVA followed by the Newman-Keuls post-hoc test were used for the comparisons with GraphPad Prism software (version 5.0, GraphPad Software, Inc., La Jolla, CA, USA). A p values of p ? 0.05 was considered significant.

Results

Sperm viability and acrosome membrane integrity determined by flow cytometry

Irradiation with a low-power laser significantly increased (p < 0.05) the percentage of live sperm cells as evaluated by flow cytometry, both in the 4 joule group (70.3 ± 4.8) and in the 6 joule group (66.9 ± 11.7) compared to the control group (57.9 ± 5.4), as shown in Fig. 1A). Similarly, low-power laser irradiation at both 4 joules (46.2 ± 2.2) and 6 joules (45.5 ± 3.3) maintained the integrity of the acrosome membrane of living cells, as assessed by flow cytometry, significantly better (p <0.05) when compared to the results obtained in the control group (38.7 ± 9.8) (Fig. 1B).

Fig 1

Representative Low Level Laser Irradiation in joules 4 and 6 and analyzed by flow cytometry.

Evaluation of sperm motility

In comparing laser irradiation at doses of 4 and 6 joules, and a non-irradiated control group, the following variables were analyzed: curvilinear speed, rectilinear speed, average value, linearity index, oscillation index, head side movement, beat frequency, mobile progressive, and straightness index. On the straightness index variable, there was a statistical difference between the control group and the group irradiated with 4 joules and also between the two treatment groups, 4 and 6 joules (p <0.05). We also found statistical differences for the variable mobile progressive when comparing the control group (30.4 ± 10.5) with the 4 joules group (43.5 ± 7.7). The straightness index result also showed statistical significance (p < 0.05) between the control group (68.7 ± 3.7) and the 4 joules group (73.1 ± 3.0), and between the 4 joules group (73.1 ± 3.0) and the 6 joules group (68.7 ± 3.8) with p < 0.05 (Fig. 2). The results of the other outcomes related to motion analysis are summarized in Table 1.

Fig 2

Evaluation of sperm movement through the Integrated Semen Analysis System in (A) Straightness Index percentage of sperm analysis presenting the non-irradiated control group and groups subjected to irradiation with low-power laser with dose 4:06 joules.…

Table 1

Results of the evaluation of sperm movement by the Integrated Semen Analysis System.

Discussion

The rationale for this study addresses the difficulties faced by farmers in improving sperm viability and the possibility of low-power laser irradiation as a solution to this problem. Studies of the use of lasers in improving sperm viability, however, have presented conflicting results. Our goal was to evaluate the effects of two different doses of irradiation by low-power laser on sperm motility and on the integrity of the plasma membrane and acrosome in cryopreserved bovine sperm. Our results point to an increase in the percentage of live sperm cells and acrosome integrity in relation to control cells when subjected to irradiation by low-power laser in two different doses (4 and 6 joules). The analysis of straightness and the percentage of cell motility show that a dose of 4 joules is more effective.

In vitro production (IVP) of bovine embryos using frozen/thawed semen is used around the world for commercial purposes. Sperm cells are exposed to a series of potential risks during cryopreservation [21]. The freeze-thaw process damages the plasma membrane and the acrosome of the sperm [22]. One of the reasons proposed to explain this variation is a change in the integrity of the sperm chromatin [21]. Cryopreservation also leads to a reduction in size of the head of the sperm as compared to fresh semen, perhaps because of damage or loss of the acrosome or overcondensation of the nuclear chromatin of sperm [6].

Cryopreservation also significantly increases the production of reactive oxygen species (ROS) in sperm. ROS have two effects on sperm function: at low concentrations they induce sperm capacitation, hyperactivation of the acrosome, and sperm-oocyte fusion and, on the other hand, excessive amounts of ROS damage DNA, inhibit sperm-oocyte fusion, and reduce sperm motility [23].

Several studies have shown that LLLI accelerates wound healing [24], enhances repair of bone defects [25], modulates the production of inflammatory mediators in joint inflammation [26], and decreases oxidative stress and muscle fatigue [27]. Others have shown the LLLI improves the activation of anti-inflammatory vasoactive peptides [15] and increases cell energy and viability [13]. The mechanism of photobiostimulation by LLLI is still unclear. It has been suggested that reactive oxygen species (ROS), which can be produced by photosensitization of endogenous chromophores such as cell cytochromes, flavins/riboflavins, and NADPH, may have an important role in this light/tissue interaction [23]. Additionally, Albuquerque-Pontes et al. [27] demonstrated that cytochrome c oxidase (complex IV of mitochondrial respiratory chain) is modulated by different wavelengths and doses of LLLT at different time-intervals.

We previously noted that LLLI with the wavelength of 660 nm, power 30 mW and doses of 4 and 6 joules was able to improve the percentage of live sperm cells evaluated by flow cytometry and maintain acrosomal membrane integrity. A dose of 4 joules also increased the percentage of mobile progressive sperm and the straightness index.

The improvement in semen quality after LLLI Has Been illustrated previously described in several species: dog [16], bovine [28], [29], rabbit [30], and turkey. [31] Using the same energy and wavelength as in previous similar studies, we show additional evidence that LLLI may result in a significant increase in the percentage of live sperm cells, integrity of acrosome membrane, and higher sperm motility.

According to Karu et al. [13], the possible primary mechanisms of light activation of spermatozoids suggest that photoacceptors are connected with oxygen metabolism and, in particular, with respiratory chains. It is important to recall that respiratory chain molecules in eukaryotic as well as prokaryotic cells are considered photoacceptors and photosignal transducers in these cells.[13]. On the other hand Lubarte et al. [32] report that LLLI inhibits calcium uptake by mitochondria and stimulates calcium to connect the vesicles of the plasma membrane of the sperm, promoting better cell maintenance.

The analysis of the percentage of live sperm cells and the acrosome membrane integrity have been used in several other studies because they are important for the diagnosis of the viability of semen after cryopreservation [7], [9], [10], [22], [33]. However, few studies with LLLI [16], [30], [31] have used these factors to analyze the improved quality of semen. It is noteworthy that these studies also differ from our study in the form of measurements used, since we have fluorescein isothiocyanato-labeled Pisum sativum agglutinin (FITC-PSA) to detect the acrosome integrity by flow cytometry. Sperm motility after LLLI has been investigated in several studies [16], [30], [31], [33], [34]. However, some studies [30], [35] showed negative outcomes regarding motility, these results may be related to the wavelength, laser power, energy density, irradiation time, as well as the experimental analysis conditions. Considering the data presented in our study and the current state of knowledge regarding the efficacy of LLLI in improving the quality of semen, we conclude that LLLI may exert beneficial effects on both the preservation of live sperm and sperm motility after cryopreservation.

Perspectives and Limitations

The low-level laser is used medically to accelerate repair processes of various types of tissue as well as to treat pain and inflammation. Pre-clinical studies have demonstrated several other possible uses. However, in vitro improvement in the quality of semen for artificial insemination has not been translated into actual practice. Possible mechanisms of low-level laser effects on the oxidative stress generated by cryopreservation include the following: (i) Superoxide anions induce hyperactivation and capacitation and are being altered by LLLI; (ii) capacitating spermatozoa produce elevated concentrations of superoxide anions themselves; and (iii) if the LLLI is capable of superoxide dismutase by removal of this ROS.

Acknowledgments

The authors would like to thank Tairana Central de Congelamento de Semên Ltda., represented by Dr. Vet. Tatiana Isaa Uherara Berton, for the donation of semen samples and technical support and suggestions. The authors would also like to thank the CAPES scholarship of Guilherme H. C. Fernandes, and UNINOVE, for all support.

Funding Statement

These authors have no support or funding to report.

Data Availability

All Data are available from the Dryad database (accession number(s) doi: 10.5061/dryad.c9k00).

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2015 Jan;30(1):235-40. doi: 10.1007/s10103-014-1653-x. Epub 2014 Sep 10.

Photobiomodulation with light-emitting diodes improves sperm motility in men with asthenozoospermia.

Ban Frangez H¹, Frangez I, Verdenik I, Jansa V, Virant Klun I.

Author information

1: Reproductive Unit, Department of Obstetrics and Gynecology, University Medical Centre Ljubljana, Slajmerjeva 3, 1000, Ljubljana, Slovenia, helena.ban.frangez@gmail.com.

Abstract

Sperm motility is an important parameter of male fertility and depends on energy consumption. Photobiomodulationwith light-emitting diode (LED) is known to stimulate respiratory chain in mitochondria of different mammalian cells. The aim of this research was to evaluate the effect of photobiomodulation with LED on sperm motility in infertile men with impaired sperm motility-asthenozoospermia. Thirty consecutive men with asthenozoospermia and normal sperm count who visited the infertility clinic of University Medial Centre Ljubljana between September 2011 and February 2012 were included in the study. Semen sample of each man was divided into five parts: one served as a non-treated (native) control and four parts were irradiated with LED of different wavelengths: (1) 850 nm, (2) 625, 660 and 850 nm, (3) 470 nm and (4) 625, 660 and 470 nm. The percentage of motile sperm and kinematic parameters were measured using a Sperm Class Analyser system following the WHO recommendations. In the non-treated semen samples, the average ratio of rapidly progressive sperms was 12% and of immotile sperm 73%. Treating with LED significantly increased the proportion of rapidly progressive sperm (mean differences were as follows: 2.83 (1.39-4.28), 3.33 (1.61-5.05), 4.50 (3.00-5.99) and 3.83 (2.31-5.36) for groups 1-4, respectively) and significantly decreased the ratio of immotile sperm (the mean differences and 95% CI were as follows: 3.50 (1.30-5.70), 4.33 (2.15-6.51), 5.83 (3.81-7.86) and 5.50 (2.98-8.02) for groups 1-4, respectively). All differences were highly statistically significant. This finding confirmed that photobiomodulation using LED improved the sperm motility in asthenozoospermia regardless of the wavelength.

2014 Jan;29(1):97-104. doi: 10.1007/s10103-013-1276-7. Epub 2013 Feb 14.

Effect of 830-nm diode laser irradiation on human sperm motility.

Salman Yazdi R¹, Bakhshi S, Jannat Alipoor F, Akhoond MR, Borhani S, Farrahi F, Lotfi Panah M, Sadighi Gilani MA.

Author information

¹Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran, r_salman_yazdi@yahoo.com.

Abstract

Sperm motility is known as an effective parameter in male fertility, and it depends on energy consumption. Low-level laser irradiation could increase energy supply to the cell by producing adenosine triphosphate. The purpose of this study is to evaluate how the low-level laser irradiation affects the human sperm motility. Fresh human semen specimens of asthenospermic patients were divided into four equal portions and irradiated by 830-nm GaAlAs laser irradiation with varying doses as: 0 (control), 4, 6 and 10 J/cm(2). At the times of 0, 30, 45 and 60 min following irradiation, sperm motilities are assessed by means of computer-aided sperm analysis in all samples. Two additional tests [HOS and sperm chromatin dispersion (SCD) tests] were also performed on the control and high irradiated groups as well. Sperm motility of the control groups significantly decreased after 30, 45 and 60 min of irradiation, while those of irradiated groups remained constant or slightly increased by passing of time. Significant increases have been observed in doses of 4 and 6 J/cm(2) at the times of 60 and 45 min, respectively. SCD test also revealed a non-significant difference. Our results showed that irradiating human sperms with low-level 830-nm diode laser can improve their progressive motility depending on both laser density and post-exposure time.

2012 Dec 26;21(4):275-85. doi: 10.5978/islsm.12-OR-16.

The Proximal Priority Theory: An Updated Technique in Low Level Laser Therapy with an 830 nm GaAlAs Laser.

Ohshiro T.

Author information

Japan Medical Laser Laboratory, Shinanomachi, Shinjuku, Tokyo, Japan.

Abstract

BACKGROUND AND AIMS:

The 830 nm GaAlAs diode laser has played an extremely active role in low level laser therapy (LLLT) since the early 1980’s. Recently, the author modified his original proximal priority laser technique (PPLT), and the current article set out to explain the improved approach and show scientific evidence for its efficacy. Laser Therapy System: The laser therapy system used was based on the GaAlAs diode (OhLase-3D1, JMLL, Japan), delivering 60 mW in continuous wave at a wavelength of 830 nm in the near infrared with a power density at the tip of the probe head of approximately 1.2 W/cm(2). Proximal Priority Laser Technique: Under the author’s PPLT concept, the brain is the control center for the body so every other part of the body is distal to the head. The main blood supply to the head is through the carotid arteries, and the deep penetration of the 830 nm beam applied to the side of the neck can involve and photoactivate the external and internal carotids, increasing the blood supply to the brain and creating a systemic parasympathetic system-mediated whole-body effect. The author has added gentle neck-stretching, trunk-stretching and his distal tissue softening approaches concomitant with the irradiation which enhance treatment efficacy.

RESULTS:

Real-time fine-plate thermography has revealed whole-body warming as a result of the PPLT, with applications including chronic pain attenuation, female infertility and functional training of paraplegic cerebral palsy patients. The warming effect had a latency from hours to days, increasing in intensity and latency with subsequent PPLT sessions. Both Doppler flowmetry and SPECT have shown increased cerebral and systemic blood flow following PPLT.

CONCLUSIONS:

PPLT is easy to deliver and offers tangible results in a large range of conditions, enhancing the efficacy of diode laser LLLT.

2012 Oct-Dec;116(4):1131-5.

Noninvasive laser therapy for outpatients with chronic inflammatory disorders of cervix.

Botez M¹, Anton C, Mircea R, Anton E.

Author information

¹Medical-Center Themis Art, Iasi., University of Medicine and Pharmacy Grigore T Popa Iasi, Faculty of Medicne.

Abstract

Chronic inflammation of the cervix can develop cervical stenosis with infertility and cervical congestion is related with the cervical cancer. We create a review of main etiological agents and methods of screening and diagnosis. We also make a brief review of modern therapeutic approach.

CONCLUSIONS:

We follow the utility of LLLT through the following aspects: evolution, indications, results of Babe?-Papanicolau screening, cytology, clinical aspects. The results of the study will allow the complex system of treatment to be used in a large category of women. We appreciate that the procedure (used in our center also) will decrease the cervical pathology, the morbidity inside the treatment, the mortality through the evolution of cervical cancer. We propose the applicability for outpatients first and then as an integrated treatment method inside hospitals for a wide access.

Laser Ther. 2012 Jul 3;21(2):97-103. doi: 10.5978/islsm.12-OR-05.

Personal Overview of the Application of LLLT in Severely Infertile Japanese Females.

Ohshiro T.

Author information

Ohshiro Clinic; Japan Medical Laser Laboratory; and Department of Plastic & Reconstructive Surgery, Keio University School of Medicine, Shinanomachi, Tokyo, Japan.

Abstract

BACKGROUND AND AIMS:

The rapidly graying population in Japan is being compounded by the rapidly-dropping birth rate. The latter is mostly due to the later ages at which women are giving birth as the marriage age has also been increasing. Giving birth at a later stage is associated with problems for both mother and child, but for older would-be mothers the greatest problem is infertility, sometimes severe. The present article will show how the application of low level laser therapy (LLLT) is a potentially effective treatment for severe infertility.

SUBJECTS AND METHODS:

Seventy-four females (average age 39.28 yr) with severe infertility in whom assisted reproductive technology (ART) had been unsuccessful (average of 9.13 yr) participated in the first part of a study from October 1996 – April 2000. LLLT was applied (830 nm, CW, GaAlAs 60 mW diode LLLT) in Ohshiro’s proximal priority technique (average 21.08 sessions) with or without other ART approaches. Based on successful outcomes, the study was then extended to March 2012, amassing a final total of 701 patients.

RESULTS:

Pregnancy was achieved in the first part of the trial in 16 patients (21.7% of 74) of whom 11 (68%) achieved successful live delivery. In the extended trial, pregnancy was achieved in 156 (22.3% of 701) with 79 live deliveries (50.1%).

CONCLUSIONS:

The use of 830 nm LLLT in the proximal priority technique at the parameters used in the present study, on its own or as an adjunct to other techniques, resulted in successful induction of pregnancy in just over 21% of severely infertile females, with a substantial number of these achieving live births. No adverse events were noted in any patient. LLLT is a pain-free and sideeffect free modality which could give hope to the increasing numbers of older females with infertility in Japan and potentially worldwide. Multinational studies are warranted.

KEYWORDS:

Artificial reproductive technology (ART); In vitro fertilization (IVF); gamete intrafallopian transfer (GIFT); insemination by donor (AID); insemination by husband (AIH); proximal priority technique; severe infertility; zygote intrafallopian transfer (ZIFT)

2012 May-Jun;33(3):469-73. doi: 10.2164/jandrol.111.013458. Epub 2011 Jul 14.

The effects of low–level laser light exposure on sperm motion characteristics and DNA damage.

Firestone RS¹, Esfandiari N, Moskovtsev SI, Burstein E, Videna GT, Librach C, Bentov Y, Casper RF.

Author information

1: Toronto Centre for Advanced Reproductive Technology, Toronto, Ontario, Canada.

Abstract

The objective of this study was to determine the effects of low–level laser light exposure on the motility of spermatozoa and on DNA damage. Thirty-three semen samples were collected for routine analysis and were classified as normospermic, oligospermic, or asthenospermic. After routine semen analysis was performed, residual semen was divided into treated and control aliquots. Treated samples were exposed to a 30-second infrared laser pulse of 50 mW/cm(2) at 905 nm, a wavelength thought to increase light-sensitive cytochrome c oxidase in the mitochondrial electron transport chain. Samples were then incubated at 37°C, and aliquots were analyzed at 30 minutes and 2 hours using computerassisted semen analysis. After incubation, 250 ?L of each sample was frozen at 280°C until DNA fragmentation analysis by flow cytometry. A significant increase in motility, most prominent in oligospermic and asthenospermic samples (85% increase), was observed 30 minutes after the treatment (P < .0001). No significant increase in DNA damage compared with control samples was observed. Significant changes in sperm motion kinetics were observed. Low–level laser light exposure appears to have a positive short-term effect on the motility of treated spermatozoa and did not cause any increase in DNA damage measured at 2 hours. We conclude that some cases of asthenospermia may be related to mitochondrial dysfunction. The implications of this study in terms of future clinical applications needs further investigation.

2003 Mar-Apr;(2):23-5.

Etiopathogenetic basis for using magnetolaser therapy in the complex treatment of male infertility.

[Article in Russian]

Iurshin VV, Sergienko NF, Illarionov VE.

Abstract

Up to 12-15% couples are infertile. The “responsibility” for infertility rests with the husband in 40-45% cases. The effects of routine drug therapy (n = 95) on a generative function are compared to those of magnetolaser therapy (n = 93) in 188 males with excretory-inflammatory infertility suffering from chronic prostatitis. Low-intensity laser infra-red radiation was used in a permanent magnetic field produced by Azor-2K unit. The magnetolaser therapy more significantly than the routine therapy raised concentration and number of mobile forms of the spermia, reduced their degenerative forms, elevated the level of serum sexual and gonadotropic hormones. In 1 year pregnancy occurred in 41.7 and 55.4% of 83 and 87 families (groups 1 and 2), respectively. The delivery took place in 35.8 and 49.7%, respectively.

Vopr Kurortol Fizioter Lech Fiz Kult. 2009 Jan-Feb;(1):25-8.

Application of low-intensity laser radiation and endotoxin-binding preparations to the treatment of female infertility

[Article in Russian]

Enukidze GG.

A total of 38 women of reproductive age (from 20 to 45 years) with chronic inflammatory gynecological diseases including 7 with primary and 9 with secondary infertility were examined by standard clinical, instrumental, and laboratory methods. In addition, variations of such important characteristics as serum endotoxin level and activity of antiendotoxin immunity were measured. The study has demonstrated participation of chronic aggression of endotoxins (of intestinal origin) in pathogenesis of the disorders of interest. Inclusion of the “antiendotoxic component” in the combined therapy allowed the efficacy of the treatment of chronic inflammation and female infertility to be greatly enhanced. It suggests the important (if not decisive) role of bacterial lipopolysacchardides in the pathogenetic mechanism underlying the problems considered in this study.

Urologiia. 2003 Mar-Apr;(2):23-5.

Etiopathogenetic basis for using magnetolaser therapy in the complex treatment of male infertility

[Article in Russian]

Iurshin VV, Sergienko NF, Illarionov VE.

Vopr Kurortol Fizioter Lech Fiz Kult. 1994 Mar-Apr;(2):24-6.

The use of laser therapy for restoring the fertilizing capacity of the ejaculate in men with a chronic genital inflammation

[Article in Russian]

Voronin IuT.

The study aimed at investigation of laser radiation effect on reproductive male function which has failed as a result of genital inflammation, versus the efficacy of routine chemotherapy. The treatment was given to 50 males of reproductive age who had been infertile for 1-12 years. 25 of them (group 1) were exposed to laser, the other 25 received standard drugs. The responses were assessed clinically and by ejaculate potency. Due to laser application clinical and ejaculate characteristics improved in the absence of side effects either on the reproductive system or the body as a whole. The author recommends laser application for treatment of ejaculate infertility in males with chronic genital inflammation.

The transforming role of biological acceptor in the reaction of a low-intensive laser irradiation.

Burlakov A B et al.

The influence of low level laser on unfertilized oocytes and spermatozoons of fish was studied. HeNe and GaAs 862 nm was used. High quality eggs (fertilization above 70%) were not influenced by laser light. The development in eggs of mean quality (fertilization 30-60%) was boosted and the best effect was found in poor quality eggs (below 20%). The fertilization rate and the reduction of the number of abnormal developing embryos was measured. After temperatural inactivation both oocytes and spermatozoons, the irradiation not only restored the movability and fertilizating capacity, but also promoted the development of inactivated oocytes after fertilization by the irradiated sparmatozoons. Red and infrared light had different effects.

Lasers Med Sci. 2005 Apr 19; [Epub ahead of print

Effect of 655-nm diode laser on dog sperm motility

Corral-Baques MI, Rigau T, Rivera M, Rodriguez JE, Rigau J.

Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Rovira i Virgili University, Reus, Spain.

Sperm motility depends on energy consumption. Low-level laser irradiation increases adenosin triphosphate (ATP) production and energy supply to the cell. The aim of this study is to analyse whether the irradiation affects the parameters that characterise dog sperm motility. Fresh dog sperm samples were divided into four groups and irradiated with a 655-nm continuous-wave diode laser with varying doses: 0 (control), 4, 6 and 10 J/cm(2). At 0, 15 and 45 min following irradiation, pictures were taken of all the groups in order to study motility with computer-aided sperm analysis (CASA). Functional tests were also performed. Average path velocity (VAP), linear coefficient (Lin) and beat cross frequency (BCF) were statistically and significantly different when compared to the control. The functional tests also showed a significant difference. At these parameters, the 655-nm continuous-wave diode laser improves the speed and linear coefficient of the sperm.

Can HeNe laser improve fertility?

Abstracts LASERmed 97; p. 138, no 112

The fertilizing potential of mouse spermatozoa was positively affected by HeNe laser in vitro. Cohen et al at the Bar-Ilan University, Israel found that the Ca2+ uptake, mainly in the mitochondria, was improved after LLLT. The results suggest that the effect of 630 nm laser irradiation is mediated through the generation of hydrogren peroxide by the spermatozoa and that this effect plays an important role in the augmentation of the sperm cell’s capacity to fertilize in vitro.