COVID-19 risk assessment and safety management operational guidelines for IVF center reopening.

PURPOSE: To promote nationwide dissemination and implementation of COVID-19 Risk Assessment and Safety Management Operational Guidelines, drawn up by SAMeR Task Force in ART centers in Argentina. Our objective is to prevent and mitigate the transmission of SARS-CoV-2 at an institutional level, while reducing the risk of infection among both physicians and patients in the context of a critical scenario in the local and Latin American healthcare system. METHODS: SAMeR Executive Committee set up a crisis committee which was made up of specialists in reproductive medicine, embryology, and healthcare management. A critical and updated review of the advances in science, documents, and recommendations released by other societies (ASRM, ESHRE, IFFS, Red LARA, societies of anesthesiologists, infectious diseases, and Occupational Safety and Health Administration-OSHA) was carried out. Likewise, there were joint meetings with the Ministry of Health of Argentina in order to draw up the guidelines. Simultaneously, ongoing medical training was carried out, thus providing added value to them, including two status surveys of the activities of the monovalent and polyvalent centers according to the country's epidemiological mapping. Four additional recommendations were made, and online training was given to healthcare workers. The aforementioned regulations were first analyzed by the healthcare providers and their practical suggestions were then added to the guidelines. RESULTS: The one-off collaborative work and the actions coordinated with the National ART Program of the Ministry of Health of Argentina resulted in the development and implementation of the present COVID-19 Risk Assessment and Safety Management Operational Guidelines at a national level. SAMeR gave recommendations for the implementation of the Management Guidelines for the center reopening, providing new safety criteria against the threat of viral contagion. A new organizational culture was promoted through the awareness of all the healthcare workers and teaching responsibility. We continue working on the compliance with a new "Code of Conduct and Commitment in Healthcare" and with workplace safety measures. We helped with transforming the theoretical knowledge into practical measures for the healthcare workers in different services, with the aim to prevent, mitigate, and/or handle contingencies at the centers/services and gamete banks, in line with the actions agreed upon with the Ministry of Health. CONCLUSIONS: As an extraordinary and uncertain event, the SARS-CoV-2 pandemic helped consolidate a volunteer-based and collaborative panel of SAMeR experts who developed the COVID-19 Risk Assessment and Safety Management Operational Guidelines as a new and readily available tool for physicians, patients, and gamete banks care. Their implementation has provided specific guidelines to minimize risk for professionals in ART clinics, as well as guaranteeing patient safety.

Pregnancy-specific {beta}-1-glycoprotein 1 and human leukocyte antigen-E mRNA in human sperm: differential expression in fertile and infertile men and evidence of a possible functional role during early development.

BACKGROUND: Mature spermatozoa contain thousands of mRNA transcripts. It has been recently shown that human sperm can deliver RNA into the oocyte, suggesting that mRNAs might have a role before or after fertilization. Human embryos express PSG1 (pregnancy-specific beta-1-glycoprotein 1) and HLA-E (human leukocyte antigen-E), molecules playing a role in implantation and early development. We compared PSG1 and HLA-E sperm mRNA levels in fertile and infertile men and we tested the hypothesis that these transcripts are selectively retained in the newly formed zygote. METHODS: Real-time RT-PCR was used to analyze sperm mRNA levels (n = 11 fertile, n = 31 infertile patients) of PSG1, HLA-E and PRM2 (protamine 2). The presence of PSG1 and HLA-E proteins was evaluated by western blot in sperm protein extracts (n = 3). Using ICSI of human sperm into hamster oocytes we evaluated the permanence of these mRNAs at different time points (n = 10 for each time) after fertilization. RESULTS: PSG1, HLA-E and PRM2 transcripts were demonstrated in ejaculated sperm. The fertile group showed significantly higher levels of PSG1 and HLA-E mRNA (both P < 0.05) than the infertile group, whereas PRM2 levels were not significantly different. However, PSG1 and HLA-E proteins were not found in ejaculated sperm. Following ICSI, PRM2 was undetectable after fertilization; conversely, PSG1 and HLA-E transcripts remained detectable for at least 24 h of zygotic development. CONCLUSIONS: We provide new evidence that indicates that human sperm deliver transcripts that may have a role in early embryo development and decreased levels of these transcripts may be associated with infertility.

beta-Microseminoprotein in human spermatozoa and its potential role in male fertility.

beta-Microseminoprotein (MSMB) is one of the most abundant proteins in human seminal plasma. The objectives of this study were: (1) to purify MSMB from seminal plasma (SP) and generate antibodies against the pure protein; (2) to investigate the interaction of MSMB with ejaculated spermatozoa and its possible effect on the spontaneous acrosome reaction (AR); and (3) to quantify MSMB content in SP and examine its relationship with the clinical sperm parameters. MSMB was purified from SP and its presence on the sperm surface was examined by indirect immunofluorescence using a specific polyclonal antibody. The effect of MSMB on the AR was evaluated using guinea pig epididymal spermatozoa as a model. MSMB quantification assay was performed with a two-site binding ELISA using two polyclonal antibodies against MSMB. MSMB was assessed in semen samples from fertile donors (controls) and subfertile patients according to World Health Organization criteria. MSMB was detected on the sperm surface and mainly localized to the acrosomal region of the head and neck. A significant spontaneous AR inhibition was observed when guinea pig epididymal spermatozoa were preincubated with MSMB. Finally, MSMB was significantly increased in subfertile patients when compared with fertile controls (P<0.02). The association of MSMB to the sperm surface, the inhibitor effect on the spontaneous AR and the increased MSMB levels found in SP in subfertile men suggests a relationship between this protein and semen quality and a possible role in the process of fertilization.

Acreditación de los laboratorios de Andrología en la República Argentina / Accreditation of Andrology laboratories in Argentina / Credenciamento dos laboratórios de Andrologia na República Argentina

La acreditación de laboratorios especializados en Andrología tiene como objetivo promover, mejorar y asegurar la calidad del servicio. Las especialidades requieren de la participación de expertos que asesoren a los organismos autónomos que efectúan las auditorías de tercera parte. El objetivo del trabajo es comunicar la experiencia de trabajo cooperativo llevado a cabo por una sociedad científica, la Sociedad Argentina de Andrología (SAA) y el Programa de Acreditación de Laboratorios (PAL) de la Fundación Bioquímica Argentina (FBA) para el aseguramiento de la calidad de la prestación bioquímica en el área andrológica. Con tal fin se firmó un convenio marco y específico de colaboración para la acreditación de laboratorios especializados en Andrología. La FBA llevaría a cabo la logística del proceso, con su plantel de auditores, aplicando como instrumento el Manual de Acreditación MA3 y la SAA proveería asesoramiento científico. Junto con las autoridades del PAL se elaboró un documento que especifica los apartados correspondientes al MA3 capítulo Nº 4 Anexo Nº 4, "Estándares para la acreditación de laboratorios especializados". Se realizó capacitación para la elaboración de la documentación y formación de los auditores en la especialidad. Esta experiencia demuestra que el trabajo cooperativo entre organizaciones permite alcanzar logros a favor de la seguridad del paciente.

Laboratory accreditation aims to promote, improve and ensure the quality of the service. The specialties require the participation of experts who advise the autonomous bodies that carry out third-party audits. The objective is to communicate the experience of cooperative work carried out by a scientific society, the Sociedad Argentina de Andrología (SAA) (Argentine Society of Andrology) and the Laboratory Certification Programme (PAL for its name in Spanish) of Fundación Bioquímica Argentina (FBA) (Argentine Biochemistry Foundation) for quality assurance of the biochemical work in the andrology area. To reach this goal, a framework and specific collaboration agreement was signed for the certification of specialized laboratories in Andrology. The FBA will carry out the logistics of the process, with its auditors' staff, applying the MA3 Accreditation Manual as an instrument, and the SAA will provide scientific advice. Together with the PAL authorities, a document was drawn to specify the sections corresponding to the MA3 chapter No. 4 Annex No. 4, "Standards for the certification of specialized laboratories". Training was carried out to prepare the documentation and the auditors in the specialty were trained as well. This experience has proven that cooperative work between organizations can achieve results favouring the patient's safety.

O credenciamento dos laboratórios visa promover, melhorar e garantir a qualidade do serviço. As especialidades requerem a participação de profissionais que assessoram os órgãos autônomos que realizam auditorias de terceiros. O objetivo é comunicar a experiência do trabalho cooperativo realizado por uma sociedade científica, a Sociedade Argentina de Andrologia (SAA) e o Programa de Credenciamento (PAL) da Fundação Bioquímica Argentina (FBA) para garantir a qualidade do trabalho bioquímico na área andrológica. Para esse fim, foi assinado um acordo-quadro e específico de cooperação para o credenciamento de laboratórios especializados em Andrologia. A FBA iria executar a logística do processo, com a sua equipe de auditores, por meio do Manual de Credenciamento MA3 como instrumento e a SAA como um instrumento e a SAA forneceria assessoramento científico. Junto com as autoridades do PAL foi elaborado um documento especificando as seções relativas ao MA3 capítulo Nº 4 Anexo Nº 4, "Normas para a credenciamento de laboratórios especializados". O treinamento foi realizado para a elaboração da documentação e formação dos auditores na especialidade. Essa experiência tem demonstrado que o trabalho cooperativo entre organizações permite atingir resultados positivos para a segurança do paciente.

Use of laptop computers connected to internet through Wi-Fi decreases human sperm motility and increases sperm DNA fragmentation.

OBJECTIVE: To evaluate the effects of laptop computers connected to local area networks wirelessly (Wi-Fi) on human spermatozoa. DESIGN: Prospective in vitro study. SETTING: Center for reproductive medicine. PATIENT(S): Semen samples from 29 healthy donors. INTERVENTION(S): Motile sperm were selected by swim up. Each sperm suspension was divided into two aliquots. One sperm aliquot (experimental) from each patient was exposed to an internet-connected laptop by Wi-Fi for 4 hours, whereas the second aliquot (unexposed) was used as control, incubated under identical conditions without being exposed to the laptop. MAIN OUTCOME MEASURE(S): Evaluation of sperm motility, viability, and DNA fragmentation. RESULT(S): Donor sperm samples, mostly normozoospermic, exposed ex vivo during 4 hours to a wireless internet-connected laptop showed a significant decrease in progressive sperm motility and an increase in sperm DNA fragmentation. Levels of dead sperm showed no significant differences between the two groups. CONCLUSION(S): To our knowledge, this is the first study to evaluate the direct impact of laptop use on human spermatozoa. Ex vivo exposure of human spermatozoa to a wireless internet-connected laptop decreased motility and induced DNA fragmentation by a nonthermal effect. We speculate that keeping a laptop connected wirelessly to the internet on the lap near the testes may result in decreased male fertility. Further in vitro and in vivo studies are needed to prove this contention.

DNA fragmentation in morphologically normal spermatozoa: how much should we be concerned in the ICSI era?

Intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility. However, there are still unanswered questions about the safety of this technique. During ICSI, only morphologically normal and motile spermatozoa are typically used to fertilize an oocyte. We recently reported that in infertile men, spermatozoa with apparently normal morphology may have DNA fragmentation. This finding consequently raised the possibility that spermatozoa with normal-shaped appearance but with DNA fragmentation could be mistakenly selected to fertilize oocytes during ICSI. This concern became more clinically significant following the subsequent finding that the presence of an increased proportion of normal spermatozoa with damaged DNA was negatively associated with embryo quality and pregnancy outcome after ICSI. Herein, we propose and discuss the hypothesis that the examination of DNA integrity in the subpopulation of highly motile (hence viable) and morphologically normal cells (and not in the total sperm population) may provide optimized information in prediction of ICSI success. More importantly, this new way of evaluation may provide reassurance about genomic normalcy and minimal risk of transmission of genetic disease and guide the development of improved methods of selection of spermatozoa with intact DNA to be used in assisted reproduction.

DNA fragmentation of normal spermatozoa negatively impacts embryo quality and intracytoplasmic sperm injection outcome.

OBJECTIVE: To evaluate DNA fragmentation in morphologically normal sperm recovered from the same sample used for intracytoplasmic sperm injection (ICSI) and to correlate DNA damage with embryo quality and pregnancy outcome. DESIGN: Prospective study. SETTING: Academic center. PATIENT(S): 36 infertile men participating in the ICSI program. INTERVENTION(S): Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick end labeling (TUNEL) assay and morphologic assessment by phase contrast. MAIN OUTCOME MEASURE(S): Simultaneous assessment of sperm morphology and DNA fragmentation by TUNEL assay was performed in the same cell, then the percentage of normal sperm with fragmented DNA (normal SFD) was correlated with embryo quality and pregnancy outcomes. RESULT(S): A highly statistically significant negative correlation was found between the percentage of normal SFD and embryo quality. This association was confirmed for the transferred embryos and for the total embryo cohort. The receiver operating characteristics curve analysis demonstrated that the percentage of normal SFD and embryo quality were statistically significant predictors of pregnancy. When the percentage of normal SFD was

Fragmentation of DNA in morphologically normal human spermatozoa.

OBJECTIVE: To evaluate DNA fragmentation in spermatozoa with normal morphological appearance. DESIGN: Prospective study. SETTING: Academic tertiary center. PATIENT(S): Fertile, subfertile, and infertile men were studied. INTERVENTION(S): Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick-end labeling assay and morphology assessment by phase contrast in the swim-up fractions. MAIN OUTCOME MEASURE(S): Simultaneous assessment of the percentage of normally shaped sperm and DNA fragmentation. RESULT(S): No DNA fragmentation was found in spermatozoa with normal morphology in any of the samples from the fertile group. In only one sample from the subfertile group did we observed normally shaped sperm cells exhibiting DNA fragmentation. However, in all the samples from the infertile group, we observed normal spermatozoa with DNA fragmentation. Spermatozoa from this late group exhibited a high proportion of DNA damage. CONCLUSION(S): In infertile men with moderate and severe teratozoospermia, the spermatozoa with apparently normal morphology present in the motile fractions after swim-up may have DNA fragmentation.

Developmental sperm contributions: fertilization and beyond.

The objective of this review was to examine the role of the various spermatozoal components suspected of actively participating in early human development. The contributions of the fertilizing spermatozoon to the oocyte include, as a minimum, the delivery of the DNA/chromatin, a putative oocyte-activating factor (OAF), and a centriole. Recent data indicate that spermatozoa may also provide the zygote with a unique suite of paternal mRNAs; some transcripts might be crucial for early and late embryonic development and deficient delivery, or aberrant transcription might contribute to abnormal development and arrest. Clinical evidence from assisted reproduction suggests that failure to complete the fertilization process, syngamy, or early cleavage might be the result of an early paternal effect. It is speculated that an abnormal release of a putative OAF and/or dysfunctions of the centrosome and cytoskeletal apparatus may mediate these effects. On the other hand, a later paternal effect resulting in embryonic failure to achieve implantation, pregnancy loss, and/or developmental abnormalities resulting from "carried over" sublethal effects may be associated with sperm nuclear/chromatin defects, including the presence of aneuploidy, genetic anomalies, DNA damage, and possibly other causes. These findings highlight the need for continuous monitoring of clinical results.

Fragmentación de ADN espermático en pacientes infértiles y su correlación con técnicas de reproducción con técnicas de reproducción asistida de alta complejidad (ICSI) / Sperm DNA fragmentation in infertile patients and its correlation with reproduction techniques with assisted reproduction of high complexity (ICSI)

La integridad del ADN es crucial para el normal desarrollo embrionario. La evaluación de la fragmentación de ADN espermático se ha propuesto como un método para predecir probabilidades de lograr un embarazo, tanto por concepción natural como por técnicas de reproducción asistida (TRA). La inyección intracitoplasmática del espermatozoide (ICSI) es la TRA de alta complejidad más utilizada en los últimos años. Sin embargo, ésta técnica es mucho más invasiva y no tiene en cuenta las características moleculares o genéticas del espermatozoide seleccionado. Lo cual podría llevar a introducir dentro del óvulo a espermatozoides con ADN dañado. Mediante la técnica de TUNEL se evaluó la fragmentación de ADN en espermatozoides de hombres infértiles que realizaban TRA por ICSI. Los valores fueron cotejados con los resultados del procedimiento. No se observaron correlaciones ni diferencias significativas entre el porcentaje de espermatozoides con fragmentación de ADN y las tasas de fertilización, calidad embrionaria o tasa de embarazo. Utilizando la evaluación simultanea de morfología espermática y fragmentación de ADN, se determinó que la mayor proporción de espermatozoides con ADN dañado eran células con morfología espermática anómala. Solo el 1,9 % de los espermatozoides con ADN fragmentado presentaban morfología normal.

Abstract: DNA integrity is crucial to normal embryonic development. Sperm DNA fragmentation evaluation has been proposed as a method to predict pregnancy, both natural conception and assisted reproductive technique (ART). Intracytoplasmatic sperm injection (ICSI) is the ART most used in the last years. However, this technique is most invasive and ignore molecular and genetics condition of selected sperm. This could lead to inject DNA damaged sperm into the egg. Sperm DNA fragmentation was evaluated by TUNEL in a fraction of the same separated sample used for ICSI. Percentage of sperm DNA damage was compared with the ICSI outcomes. There was not a statistically significant association between percentage of sperm DNA integrity and fertilization, embryo quality or pregnancy outcome. Simultaneous evaluation of sperm DNA fragmentation and morphology in the same sperm cell was used. Our data indicate that the majority of the cells showing DNA fragmentation have abnormal forms. Only 1.9% of sperm with fragmented DNA showed normal morphology. It is well known that in ICSI procedure only motile and normal sperm will be selected to injection. According this knowledge and our previous results, sperm DNA fragmentation evaluation was performed in normal spermatozoa from samples used for ICSI. Results demonstrate an association between the incidence of morphologically normal spermatozoa with fragmented DNA and poor embryo quality. In addition, a threshold was found to predict likelihood to pregnancy.

Fragmentación de ADN espermático en pacientes infértiles y su correlación con técnicas de reproducción con técnicas de reproducción asistida de alta complejidad (ICSI) / Sperm DNA fragmentation in infertile patients and its correlation with reproduction techniques with assisted reproduction of high complexity (ICSI)

La integridad del ADN es crucial para el normal desarrollo embrionario. La evaluación de la fragmentación de ADN espermático se ha propuesto como un método para predecir probabilidades de lograr un embarazo, tanto por concepción natural como por técnicas de reproducción asistida (TRA). La inyección intracitoplasmática del espermatozoide (ICSI) es la TRA de alta complejidad más utilizada en los últimos años. Sin embargo, ésta técnica es mucho más invasiva y no tiene en cuenta las características moleculares o genéticas del espermatozoide seleccionado. Lo cual podría llevar a introducir dentro del óvulo a espermatozoides con ADN dañado. Mediante la técnica de TUNEL se evaluó la fragmentación de ADN en espermatozoides de hombres infértiles que realizaban TRA por ICSI. Los valores fueron cotejados con los resultados del procedimiento. No se observaron correlaciones ni diferencias significativas entre el porcentaje de espermatozoides con fragmentación de ADN y las tasas de fertilización, calidad embrionaria o tasa de embarazo. Utilizando la evaluación simultanea de morfología espermática y fragmentación de ADN, se determinó que la mayor proporción de espermatozoides con ADN dañado eran células con morfología espermática anómala. Solo el 1,9 % de los espermatozoides con ADN fragmentado presentaban morfología normal.(AU)

Abstract: DNA integrity is crucial to normal embryonic development. Sperm DNA fragmentation evaluation has been proposed as a method to predict pregnancy, both natural conception and assisted reproductive technique (ART). Intracytoplasmatic sperm injection (ICSI) is the ART most used in the last years. However, this technique is most invasive and ignore molecular and genetics condition of selected sperm. This could lead to inject DNA damaged sperm into the egg. Sperm DNA fragmentation was evaluated by TUNEL in a fraction of the same separated sample used for ICSI. Percentage of sperm DNA damage was compared with the ICSI outcomes. There was not a statistically significant association between percentage of sperm DNA integrity and fertilization, embryo quality or pregnancy outcome. Simultaneous evaluation of sperm DNA fragmentation and morphology in the same sperm cell was used. Our data indicate that the majority of the cells showing DNA fragmentation have abnormal forms. Only 1.9% of sperm with fragmented DNA showed normal morphology. It is well known that in ICSI procedure only motile and normal sperm will be selected to injection. According this knowledge and our previous results, sperm DNA fragmentation evaluation was performed in normal spermatozoa from samples used for ICSI. Results demonstrate an association between the incidence of morphologically normal spermatozoa with fragmented DNA and poor embryo quality. In addition, a threshold was found to predict likelihood to pregnancy.(AU)