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.

Two-dimensional and three-dimensional imaging of uterus and fallopian tubes in female infertility.

Noninvasive imaging techniques play an important role in the female infertility diagnostic algorithm. In this review article, a description of the diagnostic performance and potential clinical value of both computerized tomographic virtual hysterosalpingography (CT-VHGS) and magnetic-resonance virtual hysterosalpingography (MR-VHSG) is made, describing for both techniques, to guarantee the best possible diagnostic outcome, patient preparation and normal and principal pathologic findings. Both noninvasive diagnostic imaging modalities improve the diagnostic confidence in identification of the specific cause of female infertility. These procedures are well tolerated and can be performed without tenaculum and sedation. The combined analysis of all the imaging data offers the gynecologist a wide information spectrum, enabling a better therapeutic decision. CT-VHSG and MR-VHSG are consistent diagnostic imaging modalities for the evaluation of the female reproductive system, with an excellent diagnostic performance compared with traditional diagnostic exams and allowing a comprehensive assessment of the female reproductive system.

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.

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.

Changes in DNA fragmentation during sperm preparation for intracytoplasmic sperm injection over time.

OBJECTIVE: To compare the DNA fragmentation of semen samples established by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) after incubation in polyvinylpyrrolidone (PVP) and hyaluronic acid (HA) for different time periods. DESIGN: Comparative prospective study. SETTING: Center for reproductive medicine. PATIENT(S): Twenty-seven semen samples from infertile patients. INTERVENTION(S): None. METHODS: Semen analysis and DNA fragmentation assays (TUNEL) were performed. Two groups were established: A) normal TUNEL (<20%); and B) Abnormal TUNEL (≥ 20%). TUNEL was performed in neat (T0), postgradient (TG), 1-hour postgradient (TG1), and 2-hour postgradient (TG2) samples and in TG2 samples after 0.5, 1.0, and 1.5 hours of incubation in PVP or HA. RESULT(S): TUNEL levels were significantly reduced after gradient separation compared with neat values. In group A, TUNEL levels were significantly higher in the TG2 + 1.5 hours in PVP and HA samples but did not reach abnormal levels. In group B, TUNEL levels were significantly higher in the TG2 + 1 hour in PVP and HA samples. CONCLUSION(S): Sperm DNA fragmentation significantly decreased after centrifugation gradient, regardless of the initial levels of the sample. Samples with TUNEL ≥ 20% were more susceptible to a significant increase in DNA fragmentation over time, with similar increases being observed over time for samples that were incubated in HA or PVP. These data may be relevant for sperm preparation for intracytoplasmic sperm injection.

Virtual hysterosalpingography in the diagnosis of bicornuate versus septate uterus.

OBJECTIVE: To study the value of virtual hysterosalpingography (VHSG) in the diagnosis of bicornuate vs. septate uterus. DESIGN: Retrospective study of all cases with bicornuate or septate uterus that had VHSG during the last 3 years at our center. SETTING: Private fertility center and radiology institute with university affiliations. PATIENT(S): A total of 47 patients (from more than 1,000 studies) with bicornuate uterus (n = 15) or septate uterus (n = 32) were analyzed to determine the ability of VHSG to distinguish both entities. INTERVENTION(S): Virtual hysterosalpingography was performed in patients with bicornuate or septate uterus. MAIN OUTCOME MEASURE(S): Procedure length, discomfort, amount of radiation, and ability to outline the uterine fundus. RESULT(S): We were able to clearly distinguish bicornuate from septate uterus. In all patients who underwent septoplasty the VHSG diagnosis of septate uterus was confirmed. Virtual hysterosalpingography was well tolerated by all patients; scanning took 5 seconds per patient, and the average radiation was very low (0.9 ± 0.7 mSv). CONCLUSION(S): Virtual hysterosalpingography seems to be a valuable tool in the diagnosis of common uterine anomalies.

A shared egg donor program: which is the minimum number of oocytes to be allocated?

PURPOSE: To evaluate which is the minimum number of oocytes to be allocated to each recipient in a shared egg donor program. METHODS: We analyzed 953 recipients that received at least 4 metaphase II (MII) oocytes in the period 2006-2008. We retrospectively divided the recipients according to the number of MII oocytes actually received. RESULTS: No statistically significant differences were found among the analyzed strata in clinical pregnancy rate (A:43.7%; B:45.6%; C:48.6%; D:45.5%; E:53%, P=NS) and miscarriage rate. However, the rate of top quality transferred embryos, and the embryo freezing rate were significantly higher among those recipients that received 7 or more mature eggs. CONCLUSIONS: After a large sample was analyzed, no significant differences in fresh embryo transfer outcome were encountered when a different number of oocytes was allocated. A minimum of 4 MII oocytes seems to achieve satisfactory pregnancy rates in our shared egg donor program.

Histerosalpingografía virtual por TC multidetector de 64 filas: hallazgos en 2.500 pacientes / 64-Row multidetector CT virtual hysterosalpingography: findings in 2500 patients

Objective. To depict typical features of virtual hysterosalpingography (VHSG) in infertile patients. Material and methods: 2500 VHSG performed using a 64-row MDCT were evaluated. Results: Cervical abnormalities visualized corresponded to parietal irregularities (21 percent); thickened folds (9 percent); polyps (11 percent); diverticula (5 percent); cervical strictures (8 percent), and synechiae (1.5 percent). At the level of uterine cavity, polyps (44 percent); submucous myomas (8 percent); intramural and subserosal fibroids (11 percent); synechiae (10 percent); malformations (10 percent); adenomyosis (4 percent), and cesarean section scar (3 percent). Unilateral hydrosalpinx (7 percent) and bilateral hydrosalpinx (2 percent). Tubal obstruction was reported in 6 percent of cases. The average radiation dose was 0.94 mSv. Eighty-four percent of the patients reported mild pain or no postoperative discomfort. Conclusions: The virtual hysterosalpingography allowed a proper assessment of the internal genital organs, providing useful diagnostic information on infertility and other gynecological disorders. It constitutes a virtually painless, low-dose radiation technique, besides being well tolerated by patients.

Objetivo. Ilustrar los hallazgos característicos de la histerosalpingografía virtual (HSG-V) en pacientes en estudio de infertilidad. Material y métodos: Se evaluaron 2500 HSG-V efectuadas con un equipo de TCMD de 64 filas. Resultados A nivel del cuello se visualizaron irregularidades parietales (21 por ciento); pliegues engrosados (9 por ciento); pólipos (11 por ciento); divertículos (5 por ciento); estrechez (8 por ciento; y sinequias (1,5 por ciento). A nivel de la cavidad: pólipos (44 por ciento); miomas submucosos (8 por ciento); intramurales y subserosos (11 por ciento); y sinequias (10 por ciento); malformaciones (10 por ciento); adenomiosis (4 por ciento); cicatriz de cesárea (3 por ciento). Hidrosalpinx unilateral (7 por ciento) y bilateral (2 por ciento); obstrucción tubaria en el 6 por ciento de los casos. La dosis de radiación promedio fue 0.94 mSv. El 84 por ciento de los pacientes refirieron disconfort leve o ausente. Conclusiones: La HSG-Virtual permitió una adecuada evaluación de los órganos genitales internos, brindando información diagnóstica útil en infertilidad y otros desórdenes ginecológicos. Es una técnica poco dolorosa, bien tolerada por las pacientes y con baja dosis de radiación.

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.

64-Row multidetector CT virtual hysterosalpingography.

OBJECTIVE: To illustrate the large variety of pathologies found on 64-row computed tomographic (CT) virtual hysterosalpingography (CT-VHSG) in the evaluation of the female reproductive tract in infertile patients. MATERIAL AND METHODS: We prospectively evaluated CT datasets from 209 patients with diagnosis of infertility. CT-VHSG was performed with a 64-row CT scanner using 64 x 0.625 mm(2) collimation and 0.9 mm slice thickness. A total volume of 20 mL of an iodine contrast dilution was injected into the uterine cavity. The duration of the CT scan and the grade of patient discomfort of the procedure were documented. Images were analyzed on a workstation. The CT-VHSG exams were divided in studies with normal or pathological findings; pathologies were classified according to their locations (cervical, uterine, and fallopian tube pathology). RESULTS: No complications occurred during the procedure. The mean scan time was 3.4 +/- 0.4 s; the mean patient effective dose was 2.58 +/- 0.75 mSv. In relation to the patient's discomfort evaluation, 55.5% of the patients referred no discomfort during the procedure. Cervical pathology was found in 100/209 patients, uterine pathology in 93/209 patients, and fallopian tubes pathology in 37/209 patients. CONCLUSION: A 64-row CT-VHSG provides a reliable, non-invasive alternative diagnostic technique in the infertility workup algorithm.

Clinical use of 64-row multislice computed tomography hysterosalpingography in the evaluation of female factor infertility.

OBJECTIVE: To evaluate the potential clinical value of 64-row multislice computed tomography hysterosalpingography (MSCT-H) in the evaluation of female patients with a diagnosis of infertility and compare it with the use of standard X-ray hysterosalpingography (HSG). DESIGN: Comparative prospective study. SETTING: Outpatient Imaging Diagnostic Centre, Diagnóstico Maipú, Buenos Aires, Argentina, and a university-affiliated fertility institute (Centro de Estudios en Ginecología y Reproducción, Buenos Aires, Argentina). PATIENT(S): Twenty-two female patients with a diagnosis of infertility. INTERVENTION(S): Sixty-four-row MSCT-H and conventional X-ray HSG. MAIN OUTCOME MEASURE(S): Diagnostic accuracy of MSCT-H for the evaluation of cervical, uterine, and tubal pathology. RESULT(S): We found that 64-row MSCT-H was useful in identifying female reproductive tract abnormalities, showing in the per patient analysis a sensitivity of 100% and a specificity of 85.71%. This new procedure can be performed without a tenaculum, making it well tolerated. Clinical complications did not occur in any of the patients participating in the study. CONCLUSION(S): Sixty-four-row MSCT-H is a valuable alternative diagnostic technique in the infertility workup algorithm for the evaluation of the female reproductive tract.

Virtual hysteroscopy by multidetector computed tomography.

Currently accepted techniques that evaluate the uterus and female reproductive system in the infertility workup algorithm include hysterosonography, hysteroscopy and hysterosalpingography. Based on high speed multidetector computed tomography (MDCT) which can acquire high-quality volumetric data of the pelvic region in a single brief scan, MDCT virtual hysteroscopy is proposed as a developing, non-invasive alternative diagnostic procedure for the evaluation of uterine pathology and other gynecologic disorders. Findings of the technique are illustrated.

Multidetector computed tomography virtual hysterosalpingography in the investigation of the uterus and fallopian tubes.

OBJECTIVE: To compare the efficacy of multidetector CT virtual hysterosalpingography (MDCT-VH) with conventional X-ray hysterosalpingography (HSG) in the evaluation of patients with diagnosis of infertility. METHODS: Sixty patients with diagnosis of infertility scheduled to perform a HSG, were evaluated with 16-row (n=50) and 64-row (n=10) MDCT-VH. In 35 patients the examination was performed without a tenaculum. The HSGs were carried out using standard technique. The HSG and MDCT-VH findings were compared. The duration for both examinations and patient discomfort were documented. The sensitivity and specificity of MDCT-VH for the detection of uterine pathology and tubal obstruction were calculated using the exact binomial method. Agreement between the two methods was assessed by the Cohen's kappa method (k). RESULTS: The mean duration for MDCT-VH (16 and 64-rows) was 5+/-3 min, whereas for HSG was 28+/-3. The MDCT-VH without a tenaculum was the procedure with less patient discomfort. Sensitivity, specificity and inter-method agreement for the detection of uterine pathology were 100%, 92% and k=0.92 for 16-row MDCT-VH and 100%, 100% and k=1 for 64-row MDCT-VH, respectively. Sensitivity and specificity for detection of tubal obstruction were 80% and 80% for 16-row MDCT-VH and 100% and 100% for 64-row MDCT-VH, respectively; inter-method agreement for the visualization of the tubes was k=0.54 for 16-row MDCT-VH and k=1 for 64-row MDCT-VH. CONCLUSION: This study demonstrated the feasibility of evaluating the female reproductive system by MDCT-VH. 64-Row MDCT-VH could be an alternative diagnostic technique in the infertility workup algorithm. A larger study is in progress to validate these encouraging results.

Preliminary experience with a low-cost stimulation protocol that includes letrozole and human menopausal gonadotropins in normal responders for assisted reproductive technologies.

A low-cost stimulation protocol that includes letrozole and hMG in normal responders for assisted reproductive technologies resulted in a satisfactory ongoing pregnancy rate.

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...

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