First custom next-generation sequencing infertility panel in Latin America: design and first results.

OBJECTIVE: To present the development of the first custom gene panel for the diagnosis of male and female infertility in Latin America. METHODS: We developed a next-generation sequencing (NGS) panel that assesses genes associated with infertility. The panel targeted exons and their flanking regions. Selected introns in the CFTR gene were also included. The FMR1 gene and Y chromosome microdeletions were analyzed with other recommended methodologies. An in-house developed bioinformatic pipeline was applied for the interpretation of the results. Clear infertility phenotypes, idiopathic infertility, and samples with known pathogenic variants were evaluated. RESULTS: A total of 75 genes were selected based on female (primary ovarian insufficiency, risk of ovarian hyperstimulation syndrome, recurrent pregnancy loss, oocyte maturation defects, and embryo development arrest) and male conditions (azoospermia, severe oligospermia, asthenozoospermia, and teratozoospermia). The panel designed was used to assess 25 DNA samples. Two of the variants found were classified as pathogenic and enable the diagnosis of a woman with secondary amenorrhea and a man with oligoasthenoteratozoospermia. Targeted NGS assay metrics resulted in a mean of 180X coverage, with more than 98% of the bases covered ≥20X. CONCLUSION: Our custom gene sequencing panel designed for the diagnosis of male and female infertility caused by genetic defects revealed the underlying genetic cause of some cases of infertility. The panel will allow us to develop more precise approaches in assisted reproduction.

Effect of sperm DNA fragmentation on embryo development: clinical and biological aspects.

OBJECTIVE: The aim of this study was to investigate the effect of sperm DNA fragmentation on fertilization rate, embryo development (blastulation rate), and pregnancy outcomes for ICSI cycles performed in a cohort of couples using donor eggs and to assess the remaining embryos that were not transferred or frozen for apoptotic markers. METHODS: Eighty-two women (egg recipients) were included in the study (2016) were included in the study. The recipients' mean age was 41.8±5.1 y/o (36-49), while the egg donors' mean age was 30.8±2.1 y/o (27-33). Even though donor egg cycles with frozen sperm samples are performed regularly in our center, 35 cycles were done using fresh sperm samples. The mean age of the males involved in the procedure was 40.1±5.2 y/o. Fertilization, blastulation, and pregnancy rates were assessed. The patients were divided into two groups, TUNEL <15% and ≥15%. In arrested embryos, ICC was performed to detect cleaved caspase-3, survivin, TUNEL, and DNA. The Student's t-test was used in between-group comparisons. The Mann-Whitney U-test was used to assess homogeneity. Pearson's correlation coefficient was also calculated. p<0.05 was considered statistically significant. RESULTS: This study showed that there is a negative correlation (R=-0.5) between DNA fragmentation and blastulation rate. High levels of DNA fragmentation were associated with low blastulation and pregnancy rates (per transfer); however, fertilization rate was not affected. Samples with higher levels of DNA fragmentation were associated with higher levels of DNA fragmentation in blastomeres without activating the apoptotic pathway (9.1% vs. 15.9%) (p<0.05). Blastomeres from samples with high DNA fragmentation activated the apoptotic pathway in higher levels than samples with TUNEL <15% (16.4% vs. 21.9%) (p<0.05). CONCLUSION: Sperm DNA fragmentation was negatively correlated with blastulation and pregnancy rates even in good quality oocytes. High levels of DNA damage promote embryo arrest and induce the activation of the apoptotic pathway.

Correlation between Cytoplamic Oocyte Maturation and Chromosomal Aneuploidies - Impact on fertilization, embryo quality and pregnancy.

OBJECTIVE: To establish the relationship between oocyte cytoplasmic maturation and its chromosomal status and determine the effect of this feature over the reproductive outcome in patients with sub-optimal fertilization in ART. METHODS: Fifty couples who underwent ART were selected. From nineteen patients, 22 metaphase II-MII and 18 failed-fertilized oocytes after ICSI were studied. The first polar body was collected for chromosomal analysis by aCGH. Oocytes were processed by immunocytochemistry (ICC) to determine oocyte maturation: assessment of inactive MPF status and the conformation-alignment of the metaphase plate.Other 31 couples presented sub-optimal fertilization (<50%) after ICSI, and failed-fertilized oocytes were studied by ICC. Two groups were conformed according to the main feature observed: A) cytoplasmic immaturity and sperm premature chromosome condensation and B) sperm nuclear decondensation failure with mature cytoplasm. RESULTS: Regarding MII mature oocytes, 87% had a normal metaphase plate and 84% were chromosomally normal. Contrary, immature oocytes presented abnormal metaphase plate (86%) and just 33% were euploid. In failed-fertilized oocytes: 100% of mature oocytes had a normal metaphase plate and 71% were euploid. When oocytes were cytoplasmic immature, 37% of them were normal (metaphase plate) and 50% were chromosomally normal.The global rate of aneuploidies and metaphase plate disarrangements in immature oocytes (MII+failed-fertilized) were significantly higher than mature oocytes (P<0.05).In patients with sub-optimal fertilization, the percentage of top quality embryos and pregnancy rate was significantly higher in group B (P<0.05). CONCLUSION: Oocyte cytoplasmic immaturity is related to metaphase plate anomalies and aneuploidies. Fertilized oocytes, from a cohort with sub optimal fertilization with cytoplasmic immaturity, had poorer reproductive outcomes.

Relationship Between Sperm DNA Fragmentation and Nuclear Vacuoles.

OBJECTIVE: The aim of the present study is to assess the correlation between the presence, quantity and size of nuclear vacuoles and DNA damage and chromatin status in sperm samples of men who underwent to assisted reproduction technology. METHODS: Forty six males who underwent to assisted reproductive technology (ART) were considered. According to their latest semen analysis (<3 months), were grouped into: (A) strict morphology index ≤4% (26) and (B) strict morphology index ≥14% (20). Motile sperm were selected by density gradient, and MSOME study was conducted to assess the number and size of nuclear vacuoles. DNA fragmentation (TUNEL) and DNA strand status (acridine orange) were assessed over the selected spermatozoa accordingly to their vacuole pattern. RESULTS: In group A, sperm without vacuoles (1°) have similar levels of DNA fragmentation (TUNEL) in compare to the rest of observed patterns (2°- 6°). Regarding to AO, spermatozoa with large or several vacuoles that cover more than 30-50% of the nuclear surface are AO+, but not necessarily TUNEL positive. The first three patterns of vacuoles patterns had lower levels of AO in compare to grades 4° and 6°. In group B, those sperm with one or more vacuoles greater than 30%-50% (4° and 6°), had a significant increase in TUNEL values, in relation to group 1°- 3°. Considering AO, it was found that the 4° and 6° pattern had a significantly elevated level of this marker, as same of group A (P <0.05). CONCLUSIONS: There is no relationship between the greater number and size of sperm vacuoles with high levels of DNA fragmentation in patients with severe teratozoospermia (Kruger <4%). Conversely, this relationship is evident in normal semen samples (normal morphology. Sperm selection by IMSI technique, to select non-fragmented sperm in patients with Kruger <4%, is not necessarily secured when non-vacuolated sperm is selected.

Vitrification versus slow freezing for women undergoing oocyte cryopreservation.

BACKGROUND: Oocyte cryopreservation is a technique with considerable potential in reproductive medicine, including  fertility preservation, as a way of delaying childbearing and as part of oocyte donation programs. Although the technique was relatively ineffective at first more recently numerous modifications have led to higher success rates. OBJECTIVES: To compare the effectiveness and safety of vitrification and slow freezing as oocyte cryopreservation techniques for fertility outcomes in women undergoing assisted reproduction. SEARCH METHODS: We searched electronic databases, trial registers and websites, including the Cochrane Menstrual Disorders and Subfertility Group Specialised Register of controlled trials, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE and PsycINFO (date of search 3 March 2014). SELECTION CRITERIA: Two review authors independently selected randomised controlled trials (RCTs) comparing vitrification and slow freezing for oocyte preservation in women undergoing assisted reproduction. DATA COLLECTION AND ANALYSIS: Two review authors independently extracted the data from eligible studies and assessed their risk of bias. Any disagreements were resolved by discussion or by a third review author. Data extracted included study characteristics and outcome data. The overall quality of the evidence was assessed using GRADE methods. MAIN RESULTS: Two RCTs were included in the review (106 participants). Neither study reported live birth rate. Vitrification was associated with an increased clinical pregnancy rate compared to slow freezing (RR 3.86, 95% CI 1.63 to 9.11, P = 0.002, 2 RCTs, 106 women, I(2) = 8%, moderate quality evidence). The effect of vitrification compared to slow freezing on ongoing pregnancy rates was only reported in one small study, with inconclusive findings (RR 6.07, 95% CI 0.86 to 43.04, P = 0.07, one RCT, 28 women, low quality evidence).No data were reported on adverse effects, nor were any other outcomes reported in the included trials. The evidence was limited by imprecision. We assessed the included studies as at low to unclear risk of bias as the methods were not well described. AUTHORS' CONCLUSIONS: Oocyte vitrification compared to slow freezing probably increases clinical pregnancy rates in women undergoing assisted reproduction. However, the total number of women and pregnancies were low and the imprecision is high which limits applicability. The effect on ongoing pregnancy is uncertain as data were sparse. No data were available on live births or adverse effects.

Healthy baby born after reduction of sperm DNA fragmentation using cell sorting before ICSI.

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.

Visualization of cytoskeleton components during fertilization in mammals.

The authors analyzed the cytoskeletal dynamics during human and bovine fertilization. Microtubules, microfilaments, and actin-related proteins are differently required for pronuclear migration and apposition.

Results of intracytoplasmic sperm injection in two infertile patients with abnormal organization of sperm mitochondrial sheaths and severe asthenoteratozoospermia.

OBJECTIVE: To report assisted reproduction technologies (ART) outcome and characterize severe mitochondrial sheath (MS) anomalies in two infertile asthenoteratozoospermic patients. DESIGN: Case reports. SETTING: Private IVF clinic and academic research institution. PATIENT(S): Two infertile men with asthenoteratozoospermia. INTERVENTION(S): Intracytoplasmic sperm injection (ICSI) was performed in both cases. MAIN OUTCOME MEASURE(S): Clinical and laboratory evaluation were performed and spermatozoa studied by epifluorescence microscopy and transmission electron microscopy (TEM). RESULT(S): Patient 1 had sperm with acute bendings at the level of the narrow midpieces. Mitochondria were either scarce or absent. Three ICSI embryos were transferred. A pregnancy was achieved followed by a miscarriage at the end of the first trimester. Patient 2 had sperm with very long MSs. The number of gyres was increased to more than 30. Two ICSI cycles were performed with good fertilization rates and embryo quality, but no pregnancy was achieved. CONCLUSION(S): MS defects were studied by phase-contrast, epifluorescence microscopy, and TEM that afforded a detailed view of the sperm midpiece and the topography of the whole flagellum. The results indicate that midpiece defects, while causing severe asthenozoospermia and lower fertilizing potential, may not necessarily represent negative prognostic factors in ART.

Microtubules and parental genome organisation during abnormal fertilisation in humans.

We analysed the distribution of beta-tubulins, acetylated alpha-tubulins and chromatin configuration in 113 human zygotes showing abnormal fertilisation, 16-18 h after conventional in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). After a first characterisation using phase contrast microscopy, immunofluorescence staining was performed in 67 IVF and 46 ICSI zygotes that developed one, three or more pronuclei and/or subnuclei, with or without extrusion of the second polar body. Independently of the number of pronuclei found, beta-tubulins were uniformly distributed throughout the cytoplasm of the abnormal zygotes. We did not observe any kind of microtubule alteration with respect of the ploidy level and/or its origin. The most frequent abnormal fertilisation pattern found after IVF was the presence of three or four pronuclei (74.6%). On the other hand, the presence of one pronucleus (63.0%) was the main pattern found after ICSI. No differences between the two groups were seen in terms of development of subnuclei. Anamolies detected after IVF and ICSI showed different aetiologies such as parthenogenetic activation, gynogenetic or androgenetic development, as well as digynic or diandric fertilisation.

Manejo de las parejas HIV serodiscordantes con deseos de fertilidad: la experiencia de CEGYR / Assisted conception in couples HIV serodiscordant with wish of fertility: CEGYR experience

Introducción: en los casos de parejas serodiscordantes para el virus de inmunodeficiencia adquirida (HIV) en que el varón es seropositivo y la mujer es seronegativa, hemos establecido un programa de reproducción asistida con el fin de eliminar del semen toda partícula viral infectante y poder así, inseminar a las mujeres minimizando el riesgo de transmisión de la enfermedad. Objetivo: describir nuestra experiencia en el manejo de parejas en que el hombre es seropositivo y la mujer es seronegativa, para poder satisfacer sus deseos de descendencia y a la vez proteger a la madre y al recién nacido. Material y métodos: se realizaron 12 ciclos de inseminación intrauterina con una muestra de semen previamente procesada mediante centrifugación en gradientes discontínuos (50 por ciento, 90 por ciento) de Percoll, resuspensión del sobrenadante y una técnica de swim-up migración, luego estudiada para detección viral. Resultados: se han realizado hasta el momento 12 ciclos de reproducción asistida de baja...

Alteración de mecanismos moleculares y activación abortiva en oocitos humanos durante las fallas de fecundación: aspectos biológicos / Alteration of molecular mechanisms and abortive activation in human oocytes during the failures of fertilization

Introducción: Los primeros estudios relacionados con falla de fecundación en humanos se concentraron en la citogenética del oocito. Más recientemente, los avances en imágenes digitales y microscopía de fluorescencia han permitidos investigar eventos menos conocidos como la movilidad citoplásmica de los pronúcleos masculino y femenino y los mecanismos físicos que dirigen su unión. Objetivo: Analizar cualitativa y cuantitativamente oocitos no fecundados luego de FIV e ICSI, haciendo hincapié en la organización del citoesqueleto, estado de la cromatina, organización del áster y presencia de activaciones abortivas. Materiales y Métodos: Se estudiaron 248 oocitos clasificados como "no fecundados" luego de fertilización in vitro (FIV) e inyección intracitoplásmica de espermatozoides (ICSI) 20-40 hs post inseminación o inyección. El material se procesó para inmunofluorescencia mediante la utilización de anticuerpos monoclonales para la detección de Ó y ß tubulinas y Ó tubulinas acetiladas. El material genético se estudió por tinción con Hoechst 33258 y se analizó por microscopía óptica (UV). El análisis citogenético se realizó en 69 oocitos activados luego de ICSI de acuerdo a la técnica de Tarkowski (1966). Los resultados se analizaron estadísticamente mediante el test de Chi cuadrado. Resultados y Discusión: Inmunofluorescencia: 1) FIV: La principal causa de falla de fecundación luego de FIV fue la ausencia de penetración espermática (54,9 por ciento). De los restantes oocitos estudiados, el 11,4 por ciento mostraron una falla de activación oocitaria y el 23,9 por ciento presentaron fallas en los procesos de nucleación o migración de pronúcleos. 2) ICSI: La principal causa de falla de fecundación luego de ICSI resultó ser la falla de activación oocitaria (36,5 por ciento). Un 14,6 por ciento de los oocitos remanentes detuvieron su desarrollo en la primera placa metafásica. En general, las fallas detectadas luego de FIV ó ICSI resultaron cuantitativamente diferentes. Análisis cromosómico en oocitos activados post ICSI: El estudio cromosómico permitió identificar la presencia de activaciones abortivas, incluyendo metafases III (MIII), núcleos reticulares (NR) y núcleos telofásicos (NT)

Efecto de los endometriomas, en el momento de la captación ovocitaria, sobre los resultados de la fertilización in vitro / Endometriosis effect in the moment of the oocyte aspiration on the resultas of the vitro fertilization

La infertilidad asociada con la endometriosis es frecuentemente tratada a través de una técnica de Fertilización in Vitro (FIV) Algunos autores han sugerido que la endometriosis severa puede afectar los resultados de una FIV. El propósito del siguiente estudio fue determinar el efecto de los endometriomas ováricos presentes en el momento de la captación ovocitaria sobre los resultados de la FIV