Impacto de la fragmentación del ADN espermático y la tecnología de microfluidos en los resultados de fecundación in vitro / Impact of sperm DNA fragmentation and microfluidic technology on IVF outcomes

Un diagnóstico acertado en los pacientes infértiles es clave para determinar el tratamiento de elección en un programa de reproducción asistida. En el caso del varón, el diagnóstico inicial se basa en el resultado del seminograma, el cual permite hallar problemas relacionados con la esterilidad de la pareja, pero es insuficiente para la correcta detección de la infertilidad masculina, puesto que no predice la capacidad funcional de los espermatozoides. En los últimos años, han aparecido múltiples estudios que relacionan la integridad del ADN espermático con la fertilidad. Al mismo tiempo, los laboratorios de fecundación in vitro (FIV) tienen a su alcance nuevos métodos de selección del esperma, como los microfluidos, que ayudarían a disminuir el grado de fragmentación del ADN espermático (SDF) en la muestra. En este trabajo revisamos el impacto que tienen la SDF y el uso de los dispositivos de microfluidos en los resultados de FIV con base en una selección de estudios relevantes publicados hasta febrero de 2023.(AU)

An accurate diagnosis in infertile patients is key to determine the treatment of choice in an assisted reproduction program. In the case of the male, the initial diagnosis is based on the result of the semen analysis. The semen analysis can detect problems related to the couple's infertility, but it is insufficient for the correct diagnosis of male infertility, since it does not predict the functional capacity of the spermatozoa. In recent years, multiple studies have appeared that relate sperm ADN integrity to fertility. At the same time, IVF laboratories have within their reach new methods of sperm selection, such as microfluidics, which would make it possible to reduce the degree of ADN fragmentation in the sample. In this paper we review the impact of sperm ADN fragmentation and the use of microfluidic devices on IVF outcomes based on a selection of relevant studies published up to February 2023.(AU)

Sperm Preparation with Microfluidic Sperm Sorting Chip May Improve Intracytoplasmic Sperm Injection Outcomes Compared to Density Gradient Centrifugation.

Does sperm preparation using the FERTILE PLUS™ Sperm Sorting Chip improve fertilization rates, blastocyst formation, utilization, and euploidy rates in patients undergoing intracytoplasmic sperm injection (ICSI), compared with density gradient centrifugation (DGC)? A single-cohort, retrospective data review including data from 53 couples who underwent ICSI cycles within a 12-month period. For each couple, the two closest, consecutive cycles were identified, where one used the standard technique of sperm preparation (DGC) and the subsequent used FERTILE PLUS™, therefore, couples acted as their own controls. Paired samples t-test was used to compare means for the outcomes (fertilization, blastocyst formation, utilization, and euploidy rates). Binary logistic regression analysis assessed the relationship between female age, the presence of male factor infertility, and euploidy rates. Blastocyst, utilization, and euploidy rates were significantly higher for cycles using FERTILE PLUS™ compared to DGC (76% vs 56%, p = 0.002; 60% vs 41%, p = 0.005, and 40% vs 20%, p = 0.001, respectively). Although there was an increase in fertilization rates for cycles using FERTILE PLUS™, this was not significant (72% vs 68%, p = 0.449). The euploidy rates of females ≤ 35 years were significantly increased when the FERTILE PLUS™ sperm preparation method was used, compared to the older age group (OR 2.31, p = 0.007). No significant association was found between the presence or absence of male factor infertility and euploidy rates between the two cycles. This study provides tentative evidence that the FERTILE PLUS™ microfluidic sorting device for sperm selection can improve blastocyst formation, utilization, and euploidy rates following ICSI in comparison to the DGC method.

Microfluidic sperm sorting improves ICSI outcomes in patients with increased values of Double-Strand Breaks in sperm DNA / La selección espermática mediante un dispositivo microfluídico mejora los resultados de ICSI en pacientes con valores elevados de fragmentación de cadena doble en el ADN espermático

Background: Delays in embryo kinetics, implantation failures in ICSI treatments and recurrent miscarriages have been associated with high values of Double-Strand Breaks (DSB) in sperm DNA. While conventional methods for semen preparation have been shown to be inefficient reducing DSB values, Microfluidic Sperm Sorting (MSS) devices are promising tools to reduce this damage. Objective: To study the clinical utility of an MSS device in ICSI treatments when the male partner presents increased DSB values, as compared to the use of conventional methods based on sperm motility. Methods: This retrospective cohort study included 28 infertile couples undergoing ICSI treatments. Only couples where the male partner presented increased values of DSB were included. DSB values were evaluated in semen samples by the Neutral Comet assay. Couples performed a first ICSI cycle using conventional methods for semen preparation (Density Gradients and Swim-up) and a second ICSI cycle using the ZyMōt™ICSI (formerly named FertileChip®) microfluidic device. Embryology and clinical outcomes were compared between ICSI cycles. Results: Semen parameters and the number of obtained and fertilized oocytes did not show differences between ICSI rounds. Clinical outcomes were statistically better when MSS was used: the biochemical pregnancy rate increased 28.31%; the clinical pregnancy rate increased 35.56% and the number of live births increased 35.29%, as compared to the first ICSI cycle in this group of patients. (AU)

Antecedentes: Valores elevados de fragmentación de cadena doble (DSB) en el ADN de los espermatozoides se han asociado con retrasos en la cinética embrionaria, fallos de implantación en ciclos de ICSI y con abortos de repetición. Actualmente no hay evidencias de que los métodos convencionales para la preparación del semen puedan reducir los niveles de DSB. Por el contrario, los nuevos dispositivos microfluídicos de selección espermática (MSS) han mostrado resultados prometedores en cuanto a la reducción de la fragmentación. Objetivo: Evaluar el uso de un dispositivo MSS en ciclos de ICSI donde el varón presenta niveles elevados de DSB, en comparación con el uso de métodos convencionales basados en la selección por motilidad. Métodos: Este estudio retrospectivo ha incluido a 28 parejas infértiles que han realizado ciclos de ICSI y donde se han detectado valores elevados de DSB en la muestra seminal del varón. Los niveles de DSB se han analizado mediante el test Cometa Neutro. Las parejas realizaron un primer ciclo de ICSI utilizando métodos convencionales para la preparación del semen (gradientes de densidad y Swim-up). Posteriormente, las parejas realizaron un segundo ciclo de ICSI utilizando el dispositivo microfluídico ZyMōt™ICSI (antes FertileChip®). Se han comparado los resultados de embriología y los resultados clínicos entre ambos tratamientos. Resultados: No se han encontrado diferencias entre ambos ciclos de ICSI en cuanto a parámetros seminales y el número de ovocitos obtenidos y fecundados. Los resultados clínicos fueron mejores cuando se usó el dispositivo MSS: se observó un incremento del 28,31% en la tasa de embarazo bioquímico, del 35,56% en la tasa de embarazo clínico y del 35,29% en la tasa de nacidos vivos, en comparación con el uso de métodos convencionales. (AU)

Microfluidic sperm sorting improves ICSI outcomes in patients with increased values of Double-Strand Breaks in sperm DNA.

BACKGROUND: Delays in embryo kinetics, implantation failures in ICSI treatments and recurrent miscarriages have been associated with high values of Double-Strand Breaks (DSB) in sperm DNA. While conventional methods for semen preparation have been shown to be inefficient reducing DSB values, Microfluidic Sperm Sorting (MSS) devices are promising tools to reduce this damage. OBJECTIVE: To study the clinical utility of an MSS device in ICSI treatments when the male partner presents increased DSB values, as compared to the use of conventional methods based on sperm motility. METHODS: This retrospective cohort study included 28 infertile couples undergoing ICSI treatments. Only couples where the male partner presented increased values of DSB were included. DSB values were evaluated in semen samples by the Neutral Comet assay. Couples performed a first ICSI cycle using conventional methods for semen preparation (Density Gradients and Swim-up) and a second ICSI cycle using the ZyMot™ICSI (formerly named FertileChip®) microfluidic device. Embryology and clinical outcomes were compared between ICSI cycles. RESULTS: Semen parameters and the number of obtained and fertilized oocytes did not show differences between ICSI rounds. Clinical outcomes were statistically better when MSS was used: the biochemical pregnancy rate increased 28.31%; the clinical pregnancy rate increased 35.56% and the number of live births increased 35.29%, as compared to the first ICSI cycle in this group of patients. CONCLUSIONS: The ZyMot™ICSI microfluidic device improved the reproductive outcomes in couples where the male partner presented increased DSB values, when compared to the use of conventional semen preparation techniques.

Does Microfluidic Sperm Sorting Affect Embryo Euploidy Rates in Couples with High Sperm DNA Fragmentation?

Male infertility contributes as the main factor in 30-50% of infertility cases. Conventional methods for sperm preparation have induced questioning of sperm recovery rates. The microfluidic sperm sorting (MSS) technique selects highly motile sperm with lower levels of SDF (sperm DNA fragmentation) compared to conventional sperm sorting techniques. This study aimed to determine whether utilizing this technique will reveal better embryo quality and euploidy rates in couples with repeated implantation failure (RIF) and high SDF in a new PGT-A (preimplantation genetic testing for aneuploidies) cycle. This retrospective study included couples referred to PGT-A for previous repeated ART (assisted reproductive techniques) cycle failures and with high SDF. In their new cycles, couples who accepted the technique were assigned to the MSS group, and the rest were managed with DGC (density-gradient centrifugation). Two groups were compared in terms of fertilization and euploidy rates, clinical miscarriage and live birth rates, the total number of blastocysts, and top quality blastocysts. There was no difference between the groups regarding fertilization rates, euploidy rates, clinical miscarriage, and live birth rates. The total number of blastocysts and top quality blastocysts were significantly higher in the MSS group. The MSS technique provides a higher number of top-quality blastocysts than DGC; however, neither euploidy nor live birth rates improved. Studies focusing on confounding factors to embryonic genomic status in the presence of high SDF are needed.

Microfluidic sperm selection yields higher sperm quality compared to conventional method in ICSI program: A pilot study.

An appropriate preparation technique, should be capable of isolating highquality spermatozoa for intracytoplasmic sperm injection (ICSI). The aim was to assess sperm quality parameters, DNA integrity, embryo development, and clinical outcomes using a practical and accessible Microfluidic Sperm Sorting (MSS) technique. A total of 95 ICSI cases performed using sperm samples were prepared with our MSS (group 1) or by Direct Swim Up (DSU; control) method (group 2). Both sperm quality parameters and sperm DNA fragmentation (SDF) were compared between the groups. DNA fragmentation was assessed using Sperm Chromatin Dispersion (SCD) test and fine morphology was assessed using Motile Sperm Organelle Morphology Examination (MSOME). Embryo development and clinical outcomes were compared between the groups. In the MSS group, progressive motility and the fraction of Class I sperm morphology sperm were significantly higher compared to DSU group (P < 0.01 and P < 0.001, respectively). Moreover, the rates of DNA fragmentation and immotile spermatozoa were significantly lower in MSS when compared to DSU group (P < 0.001). Also, higher rates of high-quality embryo formation (P < 0.001), implantation (P = 0.04) and pregnancy (P = 0.05) were achieved in the MSS compared to DSU groups. The MSS technique proved to be a noninvasive, disposable, easy to use, and inexpensive method for separation of high-quality spermatozoa. Both laboratory parameters and clinical outcomes were improved with application of MSS for neat sperm collection in ICSI.AbbreviationsICSI: Intracytoplasmic Sperm Injection; MSS: Microfluidic Sperm Sorting; Sperm DNA Fragmentation (SDF); SCD: Sperm Chromatin Dispersion; MSOME: Motile Sperm Organelle Morphology Examination; DGC: Density Gradient Centrifugation; DSU: Direct Swim Up; ROS: Reactive Oxygen Species; ART: Assisted Reproducetive Technology.

In vitro survival kinetics of microfluidic-sorted bovine spermatozoa.

BACKGROUND: The isolation and characterization of sperm subpopulations that can achieve fertilization is a major challenge of assisted reproduction methods. We focused on the microfluidic sperm sorter as a novel tool for collecting highly motile spermatozoa from heterogeneous semen samples. OBJECTIVES: This study primarily aims to obtain baseline information on sorted spermatozoa according to its characteristics and in vitro life span. MATERIALS AND METHODS: Frozen-thawed bull semen was subjected to microfluidic sperm sorting using diffuser-type microfluidic sperm sorter (DMSS). After sorting, samples were collected as the sorted spermatozoa and unsorted residual spermatozoa and incubated at 37°C for subsequent evaluation. The samples were assessed at different time points (0 or 1, 6, and 24 h) in terms of motility, which was measured by computer-assisted sperm analysis (CASA), membrane integrity, mitochondrial function, and adenosine triphosphate (ATP) production after sorting (0 h). To determine the characteristics and efficiency of DMSS sorting, the sorted spermatozoa were compared with samples collected using the swim-up method, a conventional method in motile sperm selection. RESULTS: A comparison between the sorted and residual spermatozoa demonstrated significantly higher motility parameters, membrane integrity, and mitochondrial function of the sorted spermatozoa until 6 h after incubation. The time course decrement of membrane and mitochondrial status were subjected to curve fitting and theoretically supported. Sperm ATP production measured immediately after sorting showed higher ATP generation of the sorted spermatozoa compared with the unsorted, frozen-thawed spermatozoa. The motility parameters and mitochondrial activity of DMSS-sorted spermatozoa were higher than the swim-up-collected spermatozoa (p < 0.05). DISCUSSION AND CONCLUSION: These results indicate that DMSS sorting can strictly select highly motile spermatozoa with the ability to maintain its membrane integrity and mitochondrial function related to ATP production. We speculate that the device that is able to sort high-quality spermatozoa can have great potential in assisted reproduction.