The importance of standardizing criteria for PGT-A interpretation of blastocysts analyzed by next-generation sequencing.

OBJECTIVE: To compare the preimplantation genetic testing for aneuploidy (PGT-A) results using the three most frequent criteria employed by preimplantation genetic laboratories and evaluate its impact on the number of euploid embryos available for transfer. METHODS: Retrospective and descriptive study including patients who underwent PGT-A between January 2018 and December 2020. Five hundred and nine PGT-A cycles and 2,079 blastocysts were analyzed by next-generation sequencing (NGS). We re-assigned the diagnosis of all blastocysts using three different criteria: strict (mosaicism thresholds from 20% to 80%), standard (from 30% to 70%) and excluding (mosaicism is not reported). We compared the euploid, aneuploid and mosaic embryos obtained in each criteria used. RESULTS: We observed PGT-A results discrepancies in 32.5% (165/509) of the cycles when the three different criteria were applied. The standard and excluding criteria showed 92 more euploid embryos (875/2,079) compared to the strict criteria (783/2,079). Evaluating the PGT-A results per cycle with the strict, standard and excluding criteria, the euploidy rates were 34.0%, 38.4% and 38.4% (p<0.001); aneuploidy rates were 59.0%, 55.8% and 61.6% (p<0.001) and mosaic rates were 7.0% and 5.8% (p<0.047), respectively. The mean number of euploid blastocysts available for transfer was 1.54±1.67 with the strict criteria, while the possibility to obtain an euploid embryo was higher if the standard or the excluding criteria were used 1.72±1.78 (p<0.001). CONCLUSIONS: This study highlights the importance of standardizing the criteria used for the interpretation of PGT-A blastocysts. We observed significant differences on PGT-A results associated solely to the criteria used.

Associations of fertility parameters with fatty acids and DNA methylation in Mexican women undergoing in vitro fertilization.

BACKGROUND: Fatty acids (FA) likely affect human fertility at multiple levels, as deviations from physiological FA profiles are obesogenic, and FA can modify DNA methylation (DNAm). Yet, the interplay of follicular fluid (FF) and serum FA with BMI and percentage body fat (PBF) in human fertility is not completely understood. Also, associations of DNAm with fertility are largely unexplored. METHODS: Reproductive parameters ranging from retrieved oocyte number to infant birth weight, were recorded in Mexican women undergoing in vitro fertilization (n = 88). Multiple regression analysis sought BMI-adjusted and age-adjusted associations. Receiver operating characteristic analysis tested for discrimination between outcomes. RESULTS: Associations of FF and serum FA were markedly distinct. While various FF FA (C16:1, C18:0, C20:2, C20:3, arachidonic acid) were significantly and inversely associated only with retrieved oocyte number, selected serum FA were associated with a broad range of pre-fertilization and post-fertilization parameters. Associations of BMI and FF FA were complex, as arachidonic acid was inversely associated with both BMI and retrieved oocyte number, while oleic acid (OA) was directly associated with BMI and PBF. Ultrasound-assessed clinical pregnancy outcome (CP) was directly associated with serum OA but inversely with its trans isomer elaidic acid (EA) and with BMI. Compounded BMI, serum EA and OA discriminated CP well (AUC = 0.74). Whole blood DNA methylation was significantly associated with and a moderate predictor (AUC = 0.66) of percent fertilized oocytes. CONCLUSIONS: Overall FF FA pool composition rather than FA identity may impact oocyte production and cellular memory of FF FA is lost as the oocyte exits the follicular environment. The contrasting associations of BMI, FF OA and arachidonic acid suggest that the control of oocyte homeostasis by FF FA is uncoupled from BMI. Further studies are warranted to assess the potential of compounding BMI with serum EA and OA to predict CP.

Evaluation of mandibular buccal shelf characteristics in the Colombian population: A cone-beam computed tomography study.

OBJECTIVE: To evaluate the mandibular buccal shelf (MBS) in terms of the angulation and bone depth and thickness according to sex, age, and sagittal and vertical skeletal patterns in a Colombian population using cone-beam computed tomography (CBCT). Accordingly, the optimal site for miniscrew insertion in this area was determined. METHODS: This descriptive, retrospective study included 64 hemi-arches of 34 patients. On CBCT images, the angulation, buccal bone depth (4 and 6 mm from the cementoenamel junction [CEJ] of MBS), and buccal bone thickness (6 and 11 mm from the CEJ of MBS) were measured at the mesial and distal roots of the mandibular first and second molars. RESULTS: There were no statistically significant differences in the angulation, depth, and thickness of MBS between male and female patients. The values for the bone around the distal root of the mandibular second molar were significantly greater than the other values. The osseous characteristics were significantly better in participants aged 16-24 years. Class III patients exhibited the best osseous characteristics, with the bone depth at 6 mm being significantly different from that in Class I and Class II patients. Although values tended to be greater in patients with low angles, the difference was not statistically significant. CONCLUSIONS: MBS provides an optimal bone surface for miniscrew insertion, with better osseous characteristics at the distal root of the mandibular second molar, 4 mm from CEJ. Adolescent patients, Class III patients, and patients with a low angle exhibit the most favorable osseous characteristics in the MBS area.

Natural-cycle in vitro fertilization (IVF) combined with in vitro maturation in infertile patients with polycystic ovarian syndrome (PCOS) requiring IVF.

OBJECTIVE: In this study, we report an experience of 59 natural-cycle IVF combined with in vitro oocyte maturation (IVF/M) cycles in patients with PCOS requiring IVF recruited based on limitations to afford a conventional IVF treatment in a 9-years period. Results of IVF/M were compared with 164 cycles of IVF in PCOS patients. MATERIAL AND METHODS: In IVF/M cycles only hCG priming was used before oocyte recovery, with in vitro maturation of immature oocytes in a commercial medium. In conventional IVF group, recombinant FSH (rFSH) and GnRH agonist/antagonist for ovarian stimulation were used. In both groups, fertilization was achieved by intracytoplasmic sperm injection (ICSI) of mature oocytes and fresh embryos transferred at day 2 or day 3. RESULTS: In all IVF/M cycles oocytes and transferable quality embryos were obtained, only in 6 IVF/M cycles mature oocytes were obtained at oocyte capture day. Clinical pregnancy rate per cycle was 39.0% vs 53.6% (p = 0.0682) and delivery rate per cycle was 30.5% vs 42.6% (p = 0.1209) in IVF/M and conventional IVF respectively. Patients with ovarian hyperstimulation syndrome (OHSS) were 0% in IVF/M vs 6.7% in conventional IVF (p = 0.0399). CONCLUSION: Our experience in a private clinic in Mexico suggests that IVF/M can be a useful initial strategy to treat PCOS patients requiring IVF with comparable delivery rates to conventional IVF and a decreased risk of ovary hyperstimulation. IVF/M may be indicated to patients with limited resources paying without insurance for their infertility treatment.

[Combined Preimplantation Genetic Testing for Aneuploidy and Monogenic Disease in a Mexican Family Affected by X-linked Hypohidrotic Ectodermal Dysplasia].

Background: Hypohidrotic ectodermal dysplasia (HED) is a genetic skin condition presenting as hypohidrosis, hypodontia, and hypotrichosis, resulting in an important burden for affected families. The most common form of HED has an X-linked inheritance and female carriers have the option of prenatal or preimplantation genetic testing (PGT) to avoid transmission of the disease. A combined PGT for a mutation in EDA gene and aneuploidies in a Mexican carrier of X-linked HED is reported. Materials and Methods: Ovarian stimulation and assisted reproduction procedures were performed in a private academic medical center. PGT for a novel c.707-1G>A (rs886039466) mutation in EDA gene and chromosomal aneuploidies was performed by massive parallel and Sanger sequencing. Results: In the first PGT, the transfer of two blastocysts did not result in a pregnancy. An accumulative stimulation approach was decided to improve pregnancy chances for a second PGT procedure. Three ovarian stimulations were performed and 10 blastocysts coming from fresh and vitrified oocytes were genetically analyzed. A single embryo transfer produced a healthy non-carrier euploid girl. Discussion: PGT combining aneuploidy and mutation analyses is an alternative for female carriers of X-linked and other Mendelian disorders in Latin-American countries. In the era of genomic and personalized medicine, medically assisted reproduction techniques, such as PGT, are shifting from only infertility to preventive genetics.

Vitamin D supplementation blocks pulmonary structural and functional changes in a rat model of perinatal vitamin D deficiency.

Whereas epidemiological data strongly link vitamin D (VD) deficiency to childhood asthma, the underlying molecular mechanisms remain unknown. Although VD is known to stimulate alveolar epithelial-mesenchymal interactions, promoting perinatal lung maturation, whether VD supplementation during this period protects against childhood asthma has not been demonstrated experimentally. Using an in vivo rat model, we determined the effects of perinatal VD deficiency on overall pulmonary function and the tracheal contraction as a functional marker of airway contractility. One month before pregnancy, rat dams were put on either a no cholecalciferol-added or a 250, 500, or 1,000 IU/kg cholecalciferol-added diet, which was continued throughout pregnancy and lactation. At postnatal day 21, offspring plasma 25(OH)D levels and pulmonary function (whole body plethysmography and tracheal contraction response to acetylcholine) were determined. 25(OH)D levels were lowest in the no cholecalciferol-supplemented group, increasing incrementally in response to cholecalciferol supplementation. Compared with the 250 and 500 IU/kg VD-supplemented groups, the no cholecalciferol-supplemented group demonstrated a significant increase in airway resistance following methacholine challenge. However, the cholecalciferol deficiency-mediated increase in tracheal contractility in the cholecalciferol-depleted group was only blocked by supplementation with 500 IU/kg cholecalciferol. Therefore, in addition to altering alveolar epithelial-mesenchymal signaling, perinatal VD deficiency also alters airway contractility, providing novel insights to asthma pathogenesis in perinatally VD-deficient offspring. Perinatal VD supplementation at 500 IU/kg appears to effectively block these effects of perinatal VD deficiency in the rat model used, providing a strong clinical rationale for effective perinatal VD supplementation for preventing childhood asthma.

Identification of a metabolic and canonical biomarker signature in Mexican HR+/HER2-, triple positive and triple-negative breast cancer patients.

Infiltrating ductal breast cancer (IDC) is the principal tumor associated-malignancy in Mexican women. In IDC, the development of intermittent hypoxia leads to an adaptive response coordinated by the transcriptional factor HIF-1α. In the present pilot, retrospective/cross-sectional study, the HIF-1α expression was analyzed in 102 tru-cut biopsies from female patients (51 ± 12 years) without previous clinical treatment and compared to 31 normal breast biopsies. The 102 IDC samples corresponded to 56% of HER2-/HR+; 8% of HER2+/HR-; 22% of triple positive (HER2+/HR+); and 14% of triple negative (TN, HER2-/HR-) subtypes. To assess HIF-1α functionality, proteomic and kinetic analysis of glycolytic as well as mitochondrial enzymes, were determined. Validation of HIF-1α as cancer biomarker was assessed by determining the contents of the commonly used biomarkers c-MYC, Ki67, and H- and K-RAS, as well as metastatic and autophagy proteins. Proteomic analysis revealed that HIF-1α, c-MYC, HER2 and COXIV contents were significantly increased in all IDC subtypes vs. normal tissue. The contents and activities of glycolytic proteins were similar between normal and IDC samples, except for HER2-/HR+ where a substantial increase of HKII was observed. Significant increase in 2OGDH and E-cadherin was detected for TN samples vs. other IDC subtypes and for normal samples. These results clearly indicated that HIF-1α + COXIV + c-MYC (+ HER2 for HER2+ subtype) may be useful to depict a breast cancer metabolic marker pattern for diagnosis, whereas the contents of HIF-1α + c-MYC + 2OGDH + E-cadherin may be an alternative useful and reliable signature for TN subtype cancer prognosis.

Robot-assisted gastroesophageal surgery: usefulness and limitations.

Robot-assisted surgery overcomes some of the limitations of traditional laparoscopic surgery. We present our experience and lessons learned in two surgical units dedicated to gastro-esophageal surgery. From June 2009 to January 2013, we performed 130 robot-assisted gastroesophageal procedures, including Nissen fundoplication (29), paraesophageal hernia repair (18), redo for failed antireflux surgery (11), esophagectomy (19), subtotal (5) or wedge (4) gastrectomy, Heller myotomy for achalasia (22), gastric bypass for morbid obesity (12), thoracoscopic leiomyomectomy (4), Morgagni hernia repair (3), lower-third esophageal diverticulectomy (1) and two diagnostic procedures. There were 80 men and 50 women with a median age of 54 years (interquartile range: 46-65). Ten patients (7.7 %) had severe postoperative complications: eight after esophagectomy (three leaks-two cervical and one thoracic-managed conservatively), one stapler failure, one chylothorax, one case of gastric migration to the thorax, one case of biliary peritonitis, and one patient with a transient ventricular dyskinesia. One redo procedure needed reoperation because of port-site bleeding, and one patient died of pulmonary complications after a giant paraesophageal hernia repair; 30-day mortality was, therefore, 0.8 %. There were six elective and one forced conversions (hemorrhage), so total conversion was 5.4 %. Median length of stay was 4 days (IQ range 3-7). Robot-assisted gastroesophageal surgery is feasible and safe, and may be applied to most common procedures. It seems of particular value for Heller myotomy, large paraesophageal hernias, redo antireflux surgery, transhiatal dissection, and hand-sewn intrathoracic anastomosis.

Contribution of robotics to minimally invasive esophagectomy.

Robot-assisted surgery has the advantages of a three-dimensional view, versatility of instruments and better ergonomics. It allows fine dissection and difficult anastomoses in deep fields. Based on our experience, we try to define what are the main contributions of robotics to minimally invasive esophagectomy. From December 2009 to July 2012, we performed 24 minimally invasive esophagectomies (9 transhiatal, 5 Ivor-Lewis and 10 three-field), 16 of them robotically (8, 5 and 3, respectively). Eighteen patients (18/24 = 75 %) received neoadjuvant therapy. Nine patients (9/24 = 37.5 %) had symptomatic complications: 4 anastomotic leaks treated conservatively, one staple failure of the gastric plasty needing reoperation, one biliary peritonitis secondary to a gangrenous cholecystitis, one intrathoracic gastric migration after the only nonresectable case, one chylothorax and one patient with major cardiopulmonary complications. The median number of lymph nodes harvested was 12 ± 7. Median length of stay was 14 ± 13.5 days. Thirty-day mortality was nil. Complications were not related to the robot itself but to the complexity of both the technique and the patient. Although we found no advantages for the use of robotics during threefield minimally invasive esophagectomy, robotic mediastinal dissection during transhiatal esophagectomy can be performed safely under direct vision. Moreover, hand-sewn robotic-assisted technique in the prone position is promising and maybe the simplest way to carry out thoracic anastomosis during Ivor-Lewis esophagectomy.

Y chromosome microdeletions in Mexican males of couples with idiopathic recurrent pregnancy loss.

AIM: To analyze the presence of Y chromosome microdeletions in males of Mexican couples with idiopathic recurrent pregnancy losses (RPL). METHODS: Seventy-one males from couples with RPL and 66 fertile males as controls were studied. DNA was isolated from peripheral lymphocytes and used to run multiplex polymerase chain reactions. Regions AZFa (sY84, sY86), AZFb (sY127, sY134) and AZFc (sY254, sY255) of the Y chromosome were analyzed according to valid guidelines recommended by the European Academy of Andrology and the European Molecular Genetics Quality Network. Also, the sequence tagged sites (STSs): DYS262 (sY67), DYS220 (sY129), DYF85S1 (sY150), DYF86S1 (sY152) and DYF87S1 (sY153) were included in order to analyze STSs previously reported as deleted. A power analysis to support our simple size was performed. RESULTS: Results show an absence of Y chromosome microdeletions in males of couples with RPL and controls with an acceptable statistical power. CONCLUSION: The study did not show an association of recurrent pregnancy loss and Y chromosome microdeletions in Mexican male partners. Based on the results, the study of Y chromosome microdeletions in couples with RPL is not considered clinically relevant.

[Intracytoplasmic morphologically selected sperm injection (IMSI) vs intracytoplasmic sperm injection (ICSI) in patients with repeated ICSI failure]. / Inyección intracitoplásmica de espermatozoides morfológicamente seleccionados (IMSI) vs inyección intracitoplásmica de espermatozoides (ICSI) en pacientes con falla repetida a ICSI.

BACKGROUND: Intracytoplasmic sperm injection (ICSI) is highly effective for the control of male factor infertility. The sperm selected for ICSI may have structural abnormalities undetectable to 400x as nuclear vacuoles, decreasing rates of pregnancy and implantation. Recent studies show that with intracytoplasmic morphologically selected sperm injection (IMSI), at higher magnification (> 6,600x), increases pregnancy and implantation rates in patients with repeated failure to ICSI. OBJECTIVE: To compare the results of the injection of selected motile sperm organelle morphology examination (MSOME) for IMSI, instead of the use of ICSI in patients with repeated failure to ICSI. PATIENTS AND METHOD: Prospective, observational cohort study. Since February 1, 2010 was administered IMSI to couples with at least two failed cycles of ICSI, and analyzed the first 30 cycles in patients under 38 years of good ovarian reserve. This study group was compared with the last 30 cycles of ICSI performed before that date, in patients with similar clinical characteristics. The IMSI was performed with a magnification of 7,676 increases for evaluation and sperm selection. RESULTS: The groups had similar clinical characteristics. The pregnancy rate with IMSI was better than with ICSI (63 vs. 50%), the difference was not significant for the size of the sample, although the trend is clear and clinically significant in favor to IMSI. The implantation rate with IMSI (44.8%) showed statistically significant differences vs. ICSI (29.7%). No significant differences in abortion rates. CONCLUSIONS: IMSI significantly improves the implantation rate in patients with repeated failure to ICSI.

Genetic screening in infertile Mexican men: chromosomal abnormalities, Y chromosome deletions, and androgen receptor CAG repeat length.

In our study, we analyzed chromosomal abnormalities, Y chromosome deletions, androgen receptor CAG repeat length and their association with defective spermatogenesis in infertile Mexican men. Eighty-two infertile patients and 40 controls were screened for karyotypic abnormalities, Y chromosome microdeletions, and CAG repeats. Nine infertile males (11%) carried chromosomal abnormalities and 10 (12.2%) presented Y chromosome microdeletions. The mean CAG repeat length was 21.6 and 20.88 base pairs in idiopathic infertile males and controls, respectively. Our results suggest that chromosomal aberrations and Y-chromosomal microdeletions are related to male infertility in Mexican men. In addition, expansion of the CAG repeat segments of the androgen receptor is not correlated with male idiopathic infertility.

A proof-of-principle study of epigenetic therapy added to neoadjuvant doxorubicin cyclophosphamide for locally advanced breast cancer.

BACKGROUND: Aberrant DNA methylation and histone deacetylation participate in cancer development and progression; hence, their reversal by inhibitors of DNA methylation and histone deacetylases (HDACs) is at present undergoing clinical testing in cancer therapy. As epigenetic alterations are common to breast cancer, in this proof-of-concept study demethylating hydralazine, plus the HDAC inhibitor magnesium valproate, were added to neoadjuvant doxorubicin and cyclophosphamide in locally advanced breast cancer to assess their safety and biological efficacy. METHODOLOGY: This was a single-arm interventional trial on breast cancer patients (ClinicalTrials.gov Identifier: NCT00395655). After signing informed consent, patients were typed for acetylator phenotype and then treated with hydralazine at 182 mg for rapid-, or 83 mg for slow-acetylators, and magnesium valproate at 30 mg/kg, starting from day -7 until chemotherapy ended, the latter consisting of four cycles of doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2 every 21 days. Core-needle biopsies were taken from primary breast tumors at diagnosis and at day 8 of treatment with hydralazine and valproate. MAIN FINDINGS: 16 patients were included and received treatment as planned. All were evaluated for clinical response and toxicity and 15 for pathological response. Treatment was well-tolerated. The most common toxicity was drowsiness grades 1-2. Five (31%) patients had clinical CR and eight (50%) PR for an ORR of 81%. No patient progressed. One of 15 operated patients (6.6%) had pathological CR and 70% had residual disease <3 cm. There was a statistically significant decrease in global 5mC content and HDAC activity. Hydralazine and magnesium valproate up- and down-regulated at least 3-fold, 1,091 and 89 genes, respectively. CONCLUSIONS: Hydralazine and magnesium valproate produce DNA demethylation, HDAC inhibition, and gene reactivation in primary tumors. Doxorubicin and cyclophosphamide treatment is safe, well-tolerated, and appears to increase the efficacy of chemotherapy. A randomized phase III study is ongoing to support the efficacy of so-called epigenetic or transcriptional cancer therapy.