Gerardo Barroso

Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients

OBJECTIVE(S) To measure vascular endothelial growth factor (VEGF), nitric oxide (NO) and leptin levels in individual ovarian follicles and to examine their relationships with perifollicular blood flow, follicular metabolic indices, and the developmental potential of the corresponding oocyte and embryo. DESIGN Prospective study. SETTING Academic, tertiary care institution. PATIENT(S) Unselected IVF patients. INTERVENTION(S) Color-pulsed Doppler analysis of perifollicular blood flow; determination of partial pressure of oxygen (pO2), partial pressure of carbon dioxide (pCO2), and pH and VEGF, leptin and NO levels in follicular fluid. MAIN OUTCOME MEASURE(S) Fertilization and day 3 embryo morphology and cleavage. RESULT(S) Fifty-five follicular fluid samples from 16 patients were studied. Mean follicular fluid levels were as follows: VEGF, 1,046+/-863.7 pg/mL (range, <63-3,332.7 pg/mL); NO3/NO2, 34.2+/-12 microM (range, 16.4-76.1 microM); and leptin, 20.1+/-12.1 ng/mL (range, 3.3-52.2 ng/mL). Vascular endothelial growth factor had a negative correlation with embryo morphology (r = -0.28, P = .01). Leptin demonstrated a negative correlation with follicular pO2 (r = -0.42, P = .005) and a positive correlation with follicular pCO2 (r = 0.36, P = .02). Follicular leptin levels correlated positively with VEGF levels (r = 0.46, P = .008) and with NO3/NO2 levels (r = 0.39, P =.006). CONCLUSION(S) Vascular endothelial growth factor, NO and leptin appear to be markers of follicular hypoxia and suboptimal embryo development. Whether fluctuations of these regulatory factors determine or reflect changes in the follicular microenvironment affecting oocyte developmental potential remains to be elucidated.

Perifollicular blood flow Doppler indices, but not follicular pO2, pCO2, or pH, predict oocyte developmental competence in in vitro fertilization

OBJECTIVE To assess the relationships among perifollicular blood flow; follicular fluid pO2, pCO2, and pH; oocyte developmental capacity; preimplantation embryo quality. DESIGN Prospective study. SETTING Academic, tertiary care institution. PATIENT(S) Unselected, gonadotropin-stimulated IVF cycles. INTERVENTION(S) Color, pulsed Doppler analysis of perifollicular blood flow, and follicular pO2, pCO2, and pH determinations of randomly designated, mapped ovarian follicles. MAIN OUTCOME MEASURE(S) Fertilization and day 3 embryo cleavage and morphology. RESULT(S) Perifollicular vascularity indices were significantly and negatively correlated with day 3 embryo cleavage. Pulsatility index and S-D ratio also were significantly and negatively correlated with follicular pO2. The same correlation was found between resistance index and the fertilization rate of preovulatory oocytes. No relationship existed between follicular metabolic analysis and fertilization or embryo quality. The resistance index had a sensitivity of 0.57 and a specificity of 0.71 for the prediction of advanced embryo cleavage status. CONCLUSION(S) Results confirm and extend previous reports demonstrating that color, pulsed Doppler ultrasound analysis of individual preovulatory follicles during IVF therapy may provide an indirect index of the developmental competence of the corresponding oocyte. Although these methods may provide means to select embryos for transfer with the highest implantation potential, the moderate predictive power showed so far may limit their clinical applicability.

HIGH FSH:LH RATIO AND LOW LH LEVELS IN BASAL CYCLE DAY 3: IMPACT ON FOLLICULAR DEVELOPMENT AND IVF OUTCOME

AbstractPurpose: To examine the impact of low basal cycle day 3 serum LH levels or a high FSH:LH ratio on IVF results. Methods: A homogeneous group of patients was analyzed as identified by normal basal cycle of follicle stimulating hormone (FSH), Luteinizing hormone (LH), and estradiol (E2) levels. High responders (high LH:FSH ratio) and low responders (high FSH or E2 levels, and women ≥42 years of age) were excluded from analysis. Only cycles stimulated with a combination of a GnRHa (luteal suppression) and pure FSH were studied. Results: Patients with low basal LH levels (<3 mIU/mL) did not differ significantly from controls in terms of response to controlled ovarian hyperstimulation but there was a clear trend toward poorer implantation and clinical pregnancy rates. On the other hand, patients with a high FSH:LH ratio (<3) had significantly fewer mature oocytes aspirated, and lower implantation and clinical pregnancy rates than patients with gonadotropin ratio ≤3. These negative effects were evident in the presence of normal basal FSH levels and after adequate matching of females age and number of embryos transferred. Conclusions: These studies highlight a negative impact of a basal cycle high FSH:LH ratio (and possibly low LH levels) on follicular development and oocyte quality in these patients subjected to pituitary down-regulation followed by pure FSH administration. A high FSH:LH ratio may be therefore used as an early biomarker of poor ovarian response.

The effect of age on the expression of apoptosis biomarkers in human spermatozoa

OBJECTIVE To evaluate the impact of age on the expression of apoptotic biomarkers in human spermatozoa. DESIGN Cross sectional, prospective study. SETTING Academic centers. PATIENT(S) Healthy volunteers with proven fertility, stratified by age (n = 25, range: 20-68 years). INTERVENTION(S) Examination of serum hormone levels and basic semen parameters, and assessment of early (plasma membrane translocation of phosphatidylserine) and late (DNA fragmentation) sperm apoptotic markers by flow cytometry (using Annexin-V binding and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). MAIN OUTCOME MEASURE(S) Apoptosis markers. RESULT(S) Advancing male age was significantly and positively correlated with Annexin-V binding results. Although not significant, there was a clear trend for increased DNA fragmentation in the older groups. The age threshold for these observations appears to be 40 years. Advancing male age was positively correlated with FSH and sex hormone-binding globulin (SHBG) levels, and negatively correlated with sperm concentration. CONCLUSION(S) Advancing male age is associated with the expression of early apoptotic markers as evidenced by significantly increased plasma membrane translocation of phosphatidylserine, as well as with a more subtle proportion of sperm carrying DNA fragmentation. This study confirmed that male age is also associated with a decline in sperm concentration.

Is the timing of implantation affected by zona pellucida micromanipulation

AbstractPurpose: Our purpose was to examine the timing ofimplantation and early embryo development following uterinetransfer of oocytes/embryos previously subjected to zonapellucida micromanipulation. Methods: A total of 68 singleton pregnancies resulting fromIVF and embryo transfer with/without micromanipulation.Patients were divided into four groups according to the typeof micromanipulation technique: assisted hatching, embryobiopsy, intracytoplasmic sperm injection, and nomicromanipulation (control group). Serial serum β-hCG levels weremeasured between 10 and 25 days after fertilization andlog-transformed. Linear regression analyses were performedand extrapolated to hCG = 10 mIU/ml (hCG10) to estimatedetectable implantation. The slopes of the regression lineswere used to estimate the rising speed of hCG, an indirectsign of embryo development. Results: There were no significant differences among groupswith respect to hCG10, the slopes or intercepts of theregression lines. Conclusions: Various oocyte/embryo microsurgicalprocedures used in ART involving zona pellucida manipulationdo not appear to affect the timing of implantation or earlyembryo development.

The apoptotic pathway in fertile and subfertile men: a case-control and prospective study to examine the impact of merocyanine 540 bodies on ejaculated spermatozoa.

OBJECTIVE To evaluate the presence of merocyanine 540 (M540) bodies and their impact on the measurement of apoptotic biomarkers in human spermatozoa. DESIGN Case-control, prospective study. SETTING Academic centers. PATIENT(S) Fertile and subfertile subjects. INTERVENTION(S) Semen samples from subfertile and fertile men, 11 per group, were analyzed for basic semen parameters and early (annexin-V binding) and late (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) sperm apoptotic biomarkers by flow cytometry. Samples were also stained with M540 to assess the presence of M540 apoptotic bodies. MAIN OUTCOME MEASURE(S) Presence of M540 apoptotic bodies. RESULT(S) Groups differed significantly in the expression of early and late apoptosis biomarkers. The percentage of M540 bodies between groups was not different. The exclusion of M540 bodies from TUNEL results did not have a significant impact on measurement in either fertile or subfertile groups. CONCLUSION(S) This study confirmed the occurrence of M540 bodies in semen and that male factor infertility is associated with an increased expression of apoptosis biomarkers. Moreover, we demonstrated that the presence of M540 bodies did not affect the quantification of apoptotic biomarkers in either group.

Sperm Flow Cytometry: Beyond Human Fertilization and Embryo Development

Male infertily is a contributing factor in up to 50% of all infertility cases, a solo cause in about 30% of them. Therefore, new and improved diagnostic methods that reduce operator variability regarding sperm defects that are not accesible by the conventional microscope scoring should be evaluated. Assisted reproductive technology (ART) has been involved in the description of alternative pathways in basic cellular functions. it is important to know that it is also related to the peri-implantatory processes that involve the sperm-oocyte interaction, cellular changes observed during fertilization, and the early and late embryo development. Several pathways have been involved at the early stages of human gametogenesis. The spermatozoon has demonstrated an intricate correlation during the fertilization process, as a transfected vector on genetic material, and as interacting with other inner components (RNAm, mitochondrial organelles, etc.). Spermatogenesis is affected by programmed death cell pathways from its packaging process through the elongated cytoplasmic structures during spermiogenesis. Flow cytometry (FC) has been an outstanding tool with the capability to select human gametes to achieve a better reproductive condition. It has been applied as a diagnostic and therapeutic tool allowing a measurable and objective selection and discrimination of spermatozoa from subfertile subjects. Using FC, we are able to know that early distribution of organelles such as mitochondria has an impact in embryo quality before genetic activation on the eight-cell stages occurs. This chapter will let the readers know the current knowledge on sperm fertilization and the relation between the embryo development and the offspring and all the tools now available for an early diagnosis and to identify therapeutic options with FC.