Magnetic activated cell sorting (MACS) with annexin V microbeads recognizes externalized phosphatidylserine (PS) residues on the surface of apoptotic spermatozoa. The successful use of this novel technique applied to a highly apoptotic semen sample before performing intracytoplasmic sperm injection (ICSI) is reported here. The use of annexin V microbeads for selecting non-apoptotic spermatozoa seems to reduce the percentage of altered cells, improving the chance of pregnancy after ICSI.
Introduction
Sperm DNA integrity is an important paternal factor that has been shown to influence fertilization rates (Marchetti et al., 2002), embryo development and pregnancy rates (Seli and Sakkas, 2005). To reduce the number of apoptotic spermatozoa, some authors postulated that the oral use of antioxidants (during 2–3 months) prior to intracytoplasmic sperm injection (ICSI) has a positive impact on the implantation and pregnancy rates (Greco et al., 2005). Other authors question this therapeutic approach and focus instead on the causes of sperm DNA breakdown (Bolle et al., 2002, Martin and Sakkas, 1998). According to Greco et al. (2005). The use of testicular spermatozoa (with lower DNA fragmentation rates) is an alternative source of cells to use for ICSI. It is postulated that the testicular spermatozoa avoid the epididymal passage preventing damage in the sperm nucleus (Ramos et al., 2004).
Recently, the use of annexin V columns, as a non-invasive method to reduce high levels of sperm apoptosis, has been reported (Said et al., 2006, Said et al., 2008). Annexin V, coupled to submicroscopical, biodegradable superparamagnetic beads, separate dead and apoptotic spermatozoa once applied on a column exposed to a strong magnetic field. This procedure is called magnetic activated cell sorting (MACS).
Within the early events of apoptosis, the phospholipid phosphatidylserine (PS), (which is normally present on the inner leaflet of the plasma membrane), becomes externalized to the outer leaflet. Annexin V has high affinity for PS (Hoogendijk et al., 2008) and when the integrity of the sperm membrane has been disturbed, annexin V can bind to it (Said et al., 2005a).
During magnetic separation, the fraction that is retained is identified as annexin V-positive while the fraction that corresponds to non-apoptotic spermatozoa (without externalized PS), that has eluted through the column is known as annexin V-negative (Miltenyi et al., 1990, Paasch et al., 2003, Said et al., 2005b). This technique allows the selection of an enriched sample of non-apoptotic spermatozoa, which could then be used for ICSI.
In the present report, the MACS annexin V-conjugated microbead technique is used to remove spermatozoa with externalized PS after sperm preparation by density gradient centrifugation (DGC). The impact of this technique on the percentage of sperm DNA fragmentation, motility and viability was evaluated. The annexin V-negative fraction obtained after magnetic separation was used for ICSI treatment in a patient with high levels of sperm apoptosis and DNA fragmentation.
The patient was a 37-year-old woman with 4 years of primary infertility due to male factor (husband was 38 years of age). She was seen at the study centre, where she showed normal day-3 FSH, LH and oestradiol concentrations; also both husband and wife had a normal karyotype. The husband had a previous history of bilateral varicocele that was surgically treated. After six cycles of intrauterine inseminations performed at another centre without pregnancy, a sperm DNA fragmentation test (TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay) was indicated and it showed 31% fragmented spermatozoa (above the normal 20% threshold) (Sergerie et al., 2005). The TUNEL assay measures the real sperm DNA damage (single- and double-strand breaks). Previous reports have given to this test a high predictive value (Alvarez and Lewis, 2008, Greco et al., 2005). Antioxidants were given for 3 months and a standard ICSI cycle was performed. Ovarian stimulation was carried out using a protocol with 300 IU of recombinant FSH (Gonal F; Merck-Serono, Buenos Aires, Argentina) and a GnRH antagonist when indicated. A total of 11 metaphase II (MII) oocytes were retrieved and microinjected by ICSI. Only five MII oocytes developed into two-pronuclei zygotes (fertilization rate 45%) and subsequently into day-2 and day-3 embryos. Embryo scoring indicated that all embryos had asymmetric blastomeres and nuclei sizes. Two poor-quality embryos were transferred on day 3 and no pregnancy ensued.
After the first ICSI attempt at another centre, a second ICSI was planned at the study institution 1 year later. During the diagnostic analysis, a semen sample showed a concentration of 30 × 106 spermatozoa/ml, an overall motility of 38% and a normal sperm morphology of 9% according to Tygerberg’s criteria (Kruger et al., 1988). Given the fact that the patient had a history of varicocele and an elevated percentage of sperm DNA fragmentation, detection of sperm apoptosis (DNA fragmentation and the apoptotic marker cleaved caspase 3) using TUNEL (Saleh et al., 2003) and immunocytochemistry (cell signalling, cleaved caspase 3 (Asp175) (5A1), rabbit mAb) (Kotwicka et al., 2008) was indicated. The TUNEL assay yielded 25% of fragmented spermatozoa and cleaved caspase 3 was present in 8% of spermatozoa (normal expected range 4.8 ± 2.9%, Kotwicka et al., 2008) (Figure 1A and B). Due to the presence of high levels of sperm fragmented DNA, the use of MACS and ICSI was discussed and agreed with the couple. After internal institutional review board and ethics committee approval at the study institute, the couple signed an informed consent and ovarian stimulation was carried out following a similar protocol to the one previously used.
The day of MACS and ICSI, the semen sample showed a concentration of 20 × 106 spermatozoa/ml, and an overall motility of 30%. After DGC, the pellet was resuspended in 2–5 ml with 1× binding buffer (BB; Miltenyi Biotec, Germany), centrifuged at 300g for 5 min and the supernatant removed. The sample was then incubated with annexin V conjugated microbeads (Miltenyi Biotec) for 30 min at 25°C during continuous agitation. After rinsing the column with BB, the sperm-bead suspension was added on top with BB and, immediately after that, the annexin V-negative fraction was obtained. To obtain the annexin V-positive fraction, the column was taken out of the magnet and 1 ml BB was added to rinse it using a plunger provided by the manufacturer (MiniMACS; Miltenyi Biotec).
Figure 1C–E and Table 1 show the effect of using MACS on the pathological semen sample. Sperm concentration, motility, viability, fragmented DNA and cleaved caspase 3 concentrations were measured in each fraction. Student’s t-test was used to compare the means between fractions. It is important to note that the MACS technique allowed a significant reduction of DNA fragmentation levels from 25% (DGC/pre-MACS) to 10% (annexin V-negative P < 0.05), also that cleaved caspase 3 concentrations were reduced but not significantly. Motility and viability increased by 15% and 13%, respectively, after using the MACS.
A total of seven oocytes were retrieved and five MII oocytes were microinjected with the annexin V-negative fraction. After 16 h, three MII oocytes developed into two pronuclei zygotes (fertilization rate 60%, compared with 45% in the previous attempt) and subsequently into day-2 (Figure 1F) and day-3 good-quality embryos. Two embryos were transferred on day 3 and a single pregnancy achieved. A healthy baby was born at term (46 XY Karyotype). The newborn showed a normal neonatal assessment.
Section snippets
Discussion
In the present report, a novel technique (MACS) was successfully applied to an apoptotic semen sample before ICSI. As far as is known, this is the first report of an ongoing pregnancy achieved with a clear reduction in the percentage of sperm DNA fragmentation.
Previous reports in healthy semen donors demonstrated that the use of microbeads (50 nm in diameter) conjugated with annexin V was an efficient method to separate intact membrane and non-apoptotic spermatozoa (Glander et al., 2002, Paasch
Acknowledgements
Special thanks are expressed to the clinical staff of CEGYR (particularly to Dr Fiszbajn) and the embryology laboratory team. We are most grateful to Dr Claudio Chillik and Dr Carlos Sueldo for their suggestions during the proof reading of the manuscript and to Dr Juan Alvarez (Institut Marques, Barcelona, Spain) for continuously sharing scientific ideas.
Is magnetic-activated cell sorting (MACS) a safe semen sample processing technique for newborns and mothers when used for semen processing prior to intracytoplasmic sperm injection (ICSI) cycles?
This retrospective multicentre cohort study involved patients undergoing ICSI cycles with either donor or autologous oocytes from January 2008 to February 2020. They were divided into two groups: those who underwent standard semen preparation (reference group) and those who had an added MACS procedure (MACS group). A total of 25,356 deliveries were assessed in the case of cycles using donor oocytes, and 19,703 deliveries from cycles using autologous oocytes. Of these, 20,439 and 15,917, respectively, were singleton deliveries. Obstetric and perinatal outcomes were retrospectively assessed. All means, rates and incidences were computed per live newborn in each study group.
There were no significant differences between the main obstetric and perinatal morbidities affecting the mothers’ and newborns’ well-being between groups using either donated or autologous oocytes. There was a significant increase in the incidence of gestational anaemia in both subpopulations (donor oocytes P = 0.01; autologous oocytes P < 0.001). However, this incidence was within the estimated prevalence for gestational anaemia in the general population. There was a statistically significant decrease in preterm (P = 0.02) and very preterm (P = 0.01) birth rates in the MACS group in cycles using donor oocytes.
The use of MACS during semen preparation before ICSI using either donor or autologous oocytes appears to be safe for the mothers’ and newborns’ well-being during pregnancy and birth. Nevertheless, a close follow-up of these parameters in the future is advised, especially concerning anaemia, in order to detect even smaller effect sizes.
Moreover, there are multiple case reports which have shown the beneficial use of MACS. A 37- year- old woman, which her male partner was responsible for infertility, could successfully bear a healthy baby boy after MACS (Rawe et al., 2010). In other cases, MACS successfully resulted in a single and twin pregnancy in couples with previous ICSI failure (Polak de Fried and Denaday, 2010).
Assisted Reproduction Techniques (ARTs) are commonly used worldwide, and sperm quality has great effect on its successfulness. Different sperm separation techniques have been developed to increase the quality of used sperms in ART. The aim is to determine the effect of sperm separation techniques including Magnetic Activated Cell Sorting (MACS), Density Gradient Centrifugation (DGC) and Swim up (SU) on pregnancy, implantation, miscarriage rates (as the primary outcomes), and also DNA fragmentation and aneuploidy (as the secondary outcomes). PubMed, EMBASE, ProQuest, Cochrane, clinicaltrials.gov, IRCT.ir, and Google Scholar databases were searched. Five RCTs were included to analyze the effects of MACS, DGC, and SU on pregnancy, implantation, and miscarriage rates. Thirty-three studies were included to analyze the effects of mentioned techniques on DNA fragmentation and aneuploidy. Our results indicated that MACS is the best sperm separation technique when compared to DGC and SU and it resulted in a higher pregnancy rate. Also, sperm separation techniques had positive effect on decreasing DNA fragmentation level and aneuploidy in sperm cells.
To investigate the effectiveness of cumulus oophorus complexes (COCs) in the physiologic selection of spermatozoa for intracytoplasmic sperm injection (ICSI).
A prospective sibling oocytes study.
Center of reproductive medicine.
Couples undergoing ICSI during 2016, females aged ≤38 years, and at least six metaphase II (MII) oocytes retrieved. Sixty patients were included in the study. Of 857 MII oocytes, 429 were allocated to the study group and were injected with the sperm selected via COCs; 428 MII oocytes were allocated as controls (C) and fertilized by conventional ICSI.
In the study group, ICSI was performed with spermatozoa that traversed the COCs in vitro.
Blastocyst/top blastocyst formation rate, fertilization rate, and oocyte utilization rate.
Oocytes injected with COC-selected spermatozoa had a significantly higher fertilization rate than the conventional ICSI group (85.31% vs. 74.77%). There were no statistically differences in cleavage and top embryo rate on day 3 between the COC-ICSI and C-ICSI groups. However, with day 5 or 6 embryos, compared with conventional ICSI, COC-ICSI significantly improved blastocyst formation rate (64.90% vs. 53.50%), blastocyst formation rate at day 5 (46.52% vs. 38.85%), top blastocyst rate (38.72% vs. 24.20%), and the usable blastocysts formation rate (62.12% vs. 46.82%). The oocyte utilization rate was improved greatly in the COC-ICSI group compared with the C-ICSI group (51.98% vs. 34.35%). Furthermore, the fertilization rate, top embryo rate on day 3, usable blastocyst rate, top blastocyst rate, and day 5 usable blastocysts rate were similar between the conventional IVF and COC-ICSI groups. Single-blastocyst transfer was performed in 82 cycles, including 44 fresh cycles and 38 frozen-thawed cycles. The cumulative embryo implantation rate in the COC-ICSI group was 64.29%, slightly higher than in the C-ICSI group (53.85%), but there was no statistical difference.
The use of COCs to select spermatozoa for ICSI appears to be effective and led to a statistically significant improvement in blastocyst development and quality.
The main aim of a sperm preparation is to maximize the number, motility, and quality of sperm from an ejaculate. Primarily sperm preparation is used for either intrauterine insemination, in vitro fertilization or intracytoplasmic sperm injection (ICSI). This article will give a broad review of current laboratory technologies and procedures, mostly focussing on the swim-up and density gradient techniques for the routine preparation of sperm from the semen. In the final part, the introduction of new sperm preparation techniques using other platforms including hyaluronan binding, magnetic activated cell separation, and microfluidics will be discussed.
While some studies have demonstrated an improved fertility outcome when using spermatozoa selected by IMSI (e.g. Antinori et al., 2008; Bartoov et al., 2003; Hazout et al., 2006), a study by Avendaño et al. (2009) also revealed that even morphologically normal spermatozoa of infertile men recovered after a swim-up procedure can still show a high incidence of DNA fragmentation. The use of monoclonal antibodies to identify apoptotic cells through magnetic cell sorting (MACS) has been proposed with the specific purpose of removing spermatozoa containing damaged DNA in the neat ejaculate or after the sperm sample has been treated with previous sperm washing-selection procedures, such as swim-up or density gradient centrifugation (DGC) (Alvares-Sedó et al., 2010; Bucar et al., 2015; De Vantery-Arrighi et al., 2009; Rawe et al., 2010). Using the MACS methodology, improvement has been reported in (i) sperm quality (De Vantery-Arrighi et al., 2009; Huang et al., 2009; Tavalaee et al., 2012), (ii) fertilization rate (Grunewald et al., 2009; Romany et al., 2010), (iii) embryo quality (Grunewald et al., 2009; Romany et al., 2014) and (iv) pregnancy rate (Alvares-Sedó et al., 2010; De Vantery-Arrighi et al., 2009; Rawe et al., 2010).
This retrospective cohort study investigated whether reproductive outcome could be improved in couples presenting with a high level of sperm DNA fragmentation (SDF) by treating the ejaculate with the magnetic cell sorting (MACS) sperm selection procedure in combination with prior density gradient centrifugation (DGC). Only men presenting with ≥30% sperm DNA in the ejaculate were included because these patients can be potentially treated with MACS to reduce the proportion of sperm presenting DNA damage. In total, 305 couples were included in this study, and from these, 216 women underwent autologous ICSI (AUTO-ICSI), whereas the remaining 89 participated in oocyte donor ICSI (DONOR-ICSI). Ejaculates were collected and DGC treated with and without MACS. Live birth and miscarriage rates resulting from ICSI observed after clinical pregnancy were determined. Sperm selection using DGC or a combination of DGC and MACS did not show any statistical difference with respect to live birth rate of couples undergoing either AUTO-ICSI or DONOR-ICSI, irrespective of whether the couples had a moderate (≥30 to <50%) or high (≥50%) level of SDF. Remarkably, there was no evidence of miscarriage in either cohort of patients (AUTO-ICSI or DONOR-ICSI) following the MACS procedure.
Desarrollar y poner a punto la técnica de dispersión de la cromatina espermática (SCD) para evaluar el grado de fragmentación del ADN en semen.
Se incluyeron 57 pacientes normozoospérmicos (OMS 2010), los cuales fueron invitados a participar del estudio luego de firmar los consentimientos correspondientes. Se evaluó el grado de fragmentación mediante técnica de SCD, de manera objetiva y subjetiva, en muestras sometidas a estrés oxidativo con H2O2 y seleccionadas mediante swim up o gradiente de densidad, así como congeladas y descongeladas.
La evaluación objetiva demuestra que la menor fragmentación se asociaría a un mayor tamaño del halo por definición, aunque se observa un aumento (p < 0,05) en el tamaño del núcleo. El tratamiento oxidante (> 100 μM de H2O2) aumenta el índice de fragmentación de forma dosis-dependiente sin afectar la viabilidad ni la motilidad espermática. Las técnicas de selección de espermatozoides mótiles evaluadas (swim up o gradiente de densidad) disminuyen el índice de fragmentación, aunque se observa cierta susceptibilidad individual. La congelación directa a –20 °C permite diferir la realización del test, independientemente del tipo de muestra.
La evaluación subjetiva de la técnica SCD es suficiente para su uso en el laboratorio clínico, siempre que se evalúe la relación halo/núcleo para la clasificación. La utilización del tratamiento oxidante permite introducir un control positivo de daño para la puesta a punto de la técnica. La selección espermática mediante técnicas de uso habitual en reproducción asistida disminuye el índice de fragmentación, observándose cierta susceptibilidad individual. La técnica SCD puede ser diferida mediante congelamiento de la muestra, lo que permite un ahorro de insumos, reactivos y tiempo de procesamiento.
To develop the sperm chromatin dispersion test (SCD) for evaluating the DNA fragmentation in semen.
57 normozoospermic (WHO 2010) patients were invited to participate in the study after signing the consents. DNA fragmentation was evaluated by SCD, objectively and subjectively, after oxidative stress with H2O2 and also in motile selected samples (swim up or density gradient), as well as in frozen and thawed samples.
The objective assessment showed that minor fragmentation is associated to a larger halo size by definition, but also a significant increase in the size of the nucleus is observed. Oxidizing treatment (> 100 μM of H2O2) increases the fragmentation index in a dose-depend way without affecting sperm viability and motility. Different techniques of motile sperm selection (swim up or density gradients) decrease the fragmentation index with an individual susceptibility. Regardless of the sample (semen, suspension or post swim up direct freezing to –20 °C allows to delay the test.
Subjective evaluation of the SCD test is sufficient for the clinical laboratory, evaluating the relation halo/nucleoid for the classification. To set up the SCD a dose-response curve with oxidizing treatment is required as a positive control of damage. The use of sperm selection methods used in assisted reproduction, decreases the fragmentation index, however individual susceptibility was observed. The SCD could be delayed by freezing the sample which allows saving of reagents and processing time.
Vanesa Rawe is the director of the Laboratory of Research and Special Studies (LABINEE) at Centro de Estudios en Ginecología y Reproducción (CEGyR), Buenos Aires, Argentina. She graduated from the University of Buenos Aires and received a PhD in cellular biology of reproduction. She obtained an advanced fellowship in reproductive sciences for postdoctoral training at the University of Pittsburgh Medical School directed by Dr Schatten. Her research has examined aspects of the biology of fertilization especially in humans. Her current interests focus on the effects of sperm and oocyte pathology on the fertilization processes and advancing technologies surrounding gamete interactions.
