Multicenter prospective study of concordance between embryonic cell-free DNA and trophectoderm biopsies from 1301 human blastocysts.

BACKGROUND: The recent identification of embryonic cell-free DNA in spent blastocyst media has opened a new era of possibilities for noninvasive embryo aneuploidy testing in assisted reproductive technologies. Yet, previous studies assessing a limited number of embryos reported variable concordance between embryonic cell-free DNA and trophectoderm biopsies, thus questioning the validity of this approach. OBJECTIVE: This study aimed to evaluate the concordance and reproducibility of testing embryonic cell-free DNA vs trophectoderm DNA obtained from the same embryo in a large sample of human blastocysts and to assess the contribution of the inner cell mass and trophectoderm to embryonic cell-free DNA released to the culture media. STUDY DESIGN: This is an interim analysis of a prospective, observational study among 8 in vitro fertilization centers in 4 continents to assess consistency between noninvasive embryo aneuploidy testing of embryonic cell-free DNA and conventional trophectoderm biopsy. The analysis included 1301 day-6/7 blastocysts obtained in 406 in vitro fertilization cycles from 371 patients aged 20-44 years undergoing preimplantation genetic testing for aneuploidy. Fresh oocytes underwent intracytoplasmic sperm injection or in vitro fertilization. No previous assisted hatching or vitrification was allowed before media collection. Individual spent blastocyst medium was collected from embryos cultured at least 40 hours from day 4. After media collection, conventional preimplantation genetic testing for aneuploidy, comprising trophectoderm biopsy and blastocyst vitrification, was performed. Embryonic cell-free DNA was analyzed blindly after embryo transfer. Inner cell mass and trophectoderm biopsies were also performed in a subset of 81 aneuploid blastocysts donated for research. RESULTS: Embryonic cell-free DNA analyses were 78.2% (866/1108) concordant with the corresponding trophectoderm biopsies. No significant differences were detected among centers ranging from 72.5% to 86.3%. Concordance rates exceeded 86% when all defined steps in the culture laboratory were controlled to minimize the impact of maternal and operator contamination. Sensitivity per center ranged from 76.5% to 91.3% and specificity from 64.7% to 93.3%. The false-negative rate was 8.3% (92/1108), and false-positive rate was 12.4% (137/1108). The 2 fertilization techniques provided similar sensitivity (80.9% vs 87.9%) and specificity (78.6% vs 69.9%). Multivariate analysis did not reveal any bias from patient clinical background, ovarian stimulation protocols, culture conditions, or embryo quality on testing accuracy of concordance. Moreover, concordances of embryonic cell-free DNA with trophectoderm and inner cell mass suggest that the embryonic cell-free DNA originates from both compartments of the human embryo. CONCLUSION: Noninvasive analysis of embryonic cell-free DNA in spent blastocyst culture media demonstrates high concordance with trophectoderm biopsy results in this large multicenter series. A noninvasive approach for prioritizing embryo euploidy offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population.

Involvement of the ANGPTs/Tie-2 system in ovarian hyperstimulation syndrome (OHSS).

Ovarian hyperstimulation syndrome (OHSS) is a disorder associated with ovarian stimulation. OHSS features are ovarian enlargement with fluid shifting to the third space. Disturbances in the vasculature are considered the main changes that lead to OHSS. Our aim was to analyze the levels of angiopoietins 1 and 2 (ANGPT1 and 2) and their soluble and membrane receptors (s/mTie-2) in follicular fluid (FF) and in granulosa-lutein cells culture (GLCs) from women at risk of developing OHSS. We also evaluated the effect of ANGPT1 on endothelial cell migration. In ovaries from an OHSS rat model, we analyzed the protein concentration of ANGPTs, their mTie-2 receptor, and platelet-derived growth factor PDGF-B, -D and PDGFR-ß. ANGPT1 levels were increased in both FF and GLCs from women at risk of OHSS. Incubation of these FF with an ANGPT1 neutralizing antibody decreased endothelial cell migration. In the ovaries of OHSS rat model, mTie-2 protein levels increased and PDGF-B and -D decreased. In summary, these results suggest that ANGPT1 could be another mediator in the development of OHSS.

Inhibition of cyclooxygenase-2 (COX-2) by meloxicam decreases the incidence of ovarian hyperstimulation syndrome in a rat model.

OBJECTIVE: To investigate the effects of selective cyclooxygenase-2 (COX-2) inhibition on the ovarian hyperstimulation syndrome (OHSS) in an experimental model. DESIGN: Controlled laboratory study. SETTING: University-affiliated fertility center. ANIMAL(S): Female Wistar rats. INTERVENTION(S): Female Wistar rats (22 days old) were divided into four groups: group 1 (control group; n = 10) received 0.1 mL of intraperitoneal (IP) saline from days 22-26; group 2 (mild-stimulated group; n = 10) received 10 IU of pregnant mare serum gonadotropin (PMSG) on day 24 and 10 IU of hCG 48 hours later (day 26); group 3 (OHSS group; n = 10) was given 10 IU of PMSG for 4 consecutive days from day 22 and 30 IU hCG on the fifth day to induce OHSS; group 4 was treated the same as group 3, but received 2 muL (15 mg/mL) of meloxicam 2 hours before the PMSG injection for 4 consecutive days, and 2 hours before the hCG injection on the fifth day. All groups were killed on day 26. MAIN OUTCOME MEASURE(S): Number of antral and luteinized follicles, ovarian weight, semiquantitative vascular endothelial growth factor (VEGF) and COX-2 immunohistochemistry. RESULT(S): There were no differences in the ovarian weight between groups 1 and 2. Group 3 showed significantly increased ovarian weight that was suppressed, in group 4, by meloxicam. There was no difference in the number of antral follicles among the four groups. In the mild-stimulated and OHSS groups, the granulosa cells (GC) of preovulatory follicles and the stromal cells showed intense VEGF immunoreactivity. The ovaries from the meloxicam-treated group showed less immunoreactivity than the OHSS group, indicating diminished VEGF expression associated with meloxicam treatment. Group 3 (OHSS group) showed increased COX-2 immunoreactivity that was diminished in the meloxicam-treated group. Meloxicam treatment did not affect the hormone-induced increase in serum E(2) levels seen in OHSS rats. CONCLUSION(S): Our results in a rat model suggest that meloxicam has a beneficial effect on OHSS by reducing the increases in ovarian weight and VEGF expression associated with OHSS. These effects may be mediated by the COX-2 inhibitory capacity of meloxicam.

Significance of ovarian macrophages in the follicular aspirates from ART patients.

PURPOSE: To evaluate the percentages of macrophages present in granulosa cells (GC) cultures from patients with different responses to the hyperstimulation, in relation to the percentages of apoptotic cells (ApC), as well as to the release of cytokines. METHODS: We studied 42 patients: 12 Hyporesponders, (with < or =4 follicles), 15 Normoresponders, (5-14 follicles), and 15 Hyperresponders, (> or =15 follicles). In GC cultures percentages of macrophages and ApC were counted and, in the conditioned media, cytokines were measured. RESULTS: Percentages of macrophages were significantly higher in GC cultures from Hyporesponders compared with Hyperresponders patients. Also, the percentages of ApC cells were the highest in Hyporesponders. On the contrary, cytokines concentrations were the lowest in this group. CONCLUSIONS: The low ovarian response is probably due to the decreased angiogenesis, which in turn produces increased apoptosis and decreased production of cytokines. The increased percentage of macrophages could be related to increased frequency of apoptotic cells.

Direct injection of vascular endothelial growth factor into the ovary of mice promotes follicular development.

OBJECTIVE: To investigate the effects of an ovarian injection of vascular endothelial growth factor (VEGF) on antral follicle development, neoangiogenesis, and apoptosis. DESIGN: Controlled laboratory study. SETTING: University-affiliated fertility center. ANIMAL(S): Balb/c female mice (n = 32) were studied. INTERVENTION(S): Mice were divided into four groups: control group (C) n = 6, no treatment; hyperstimulated group (HS), n = 8, ovaries were stimulated with 7.5 IU pregnant mare serum gonadotropin (PMSG) and 10 IU of hCG; VEGF group (V), n = 8, injected with 0.1 mL of VEGF (0.2 microg) in each ovary; V+HS, n = 8 injected with VEGF and 2 weeks later hyperstimulated. MAIN OUTCOME MEASURE(S): Number of antral and luteinized follicles, number of vessels, and percentage of Bcl-2-positive cells. RESULT(S): The number of antral follicles with VEGF was higher than in the C and HS groups (16.0 +/- 2.5 vs. 6.0 +/- 0.9 and 11.3 +/- 0.6, respectively, p<0.005). All treatments significantly increased the number of vessels (C: 5.0 +/- 0.5 vs. V: 20.0 +/- 4.8, p<0.005 and V+HS: 22.2 +/- 1.2, p<0.01), as well as increased Bcl-2-positive cells compared to controls (C: 0; V: 11.8 +/- 3.5, p<0.005; V+HS: 12.5 +/- 3.7, p<0.005). CONCLUSION(S): Our findings demonstrated that a direct injection of VEGF into the mouse ovary results in the development of an enhanced vascular network promoting follicular development and diminishing apoptosis.

Estimación del peso fetal en nuestra población: la precisión de los modelos originados en otras poblaciones / Estimation of fetal weight in our population: the precision of the models originate in other populations

Una de las principales variables en la morbimortalidad perinatal es el peso al nacer. El objetivo del siguiente trabajo es determinar la precisión de los modelos ecográficos actualmente más utilizados en la estimación del peso fetal en nuestra población. Se examinaron en forma retrospectiva durante un período de 36 meses un total de 200 embarazadas con gestaciones únicas que tuvieron un parto dentro de los 7 días posteriores a un examen ecográfico. El Diámetro Biparietal (DBP), la Longitud Femoral (LF) y la Circunferencia Abdominal (CA) fueron medidos en todos los casos. La estimación del peso fetal fue efectuada por cuatro métodos diferentes basados en el modelo de regresión lineal que utilizaron respectivamente el CA; CA-DBP; CA y LF; YDBP, LF y CA. Los resultados fueron comparados con el peso al nacer al momento del parto. El método de Shepard fue el que mostró mayor precisión en la estimación del peso fetal: Error porcentual: - 0,86; Desviación Estándar: 9,41. Los otros modelos presentaron diferentes variaciones para distintos rangos de peso al nacer. Todas las fórmulas demostraron una subestimación significativa a partir del séptimo día de intervalo entre el examen ecográfico y el momento del parto

Estimación del peso fetal en nuestra población: la precisión de los modelos originados en otras poblaciones / Estimation of fetal weight in our population: the precision of the models originate in other populations

Una de las principales variables en la morbimortalidad perinatal es el peso al nacer. El objetivo del siguiente trabajo es determinar la precisión de los modelos ecográficos actualmente más utilizados en la estimación del peso fetal en nuestra población. Se examinaron en forma retrospectiva durante un período de 36 meses un total de 200 embarazadas con gestaciones únicas que tuvieron un parto dentro de los 7 días posteriores a un examen ecográfico. El Diámetro Biparietal (DBP), la Longitud Femoral (LF) y la Circunferencia Abdominal (CA) fueron medidos en todos los casos. La estimación del peso fetal fue efectuada por cuatro métodos diferentes basados en el modelo de regresión lineal que utilizaron respectivamente el CA; CA-DBP; CA y LF; YDBP, LF y CA. Los resultados fueron comparados con el peso al nacer al momento del parto. El método de Shepard fue el que mostró mayor precisión en la estimación del peso fetal: Error porcentual: - 0,86; Desviación Estándar: 9,41. Los otros modelos presentaron diferentes variaciones para distintos rangos de peso al nacer. Todas las fórmulas demostraron una subestimación significativa a partir del séptimo día de intervalo entre el examen ecográfico y el momento del parto (AU)