Fernando Bonilla-Musoles

The follicular and endocrine environment in women with endometriosis: local and systemic cytokine production

OBJECTIVE To assess the endocrine, paracrine, and autocrine milieu in patients with endometriosis on the basis of the measurement of several cytokines in serum and follicular fluid (FF) and in vitro culture of granulosa luteal cells. DESIGN Case-control study. SETTING In vitro fertilization program at the Instituto Valenciano de Infertilidad. PATIENT(S) Twenty patients with laparoscopically documented endometriosis and 18 controls. Fifteen subjects were studied in a natural cycle and 23 were investigated in a stimulated cycle while undergoing IVF. INTERVENTION(S) Individual follicle aspiration, oocyte isolation, FF storage, and preparation of luteinized granulosa cell cultures. Diagnostic laparoscopy in natural cycles. MAIN OUTCOME MEASURE(S) Serum (day of ovum pick-up or laparoscopy) and FF measurement of interleukin (IL)-1beta, IL-6, and vascular endothelial growth factor (VEGF). Secretion of IL-1beta, IL-6, and VEGF in the cell-conditioned medium. Results were compared between patients with endometriosis and controls. RESULT(S) Interleukin-6 levels in serum were increased in the natural cycles of patients with endometriosis and modulated by ovarian stimulation, showing a significant decrease in hMG- and FSH-stimulated cycles and a significant increase after hCG administration. In addition, IL-6 levels were increased in the FF of patients with endometriosis and released in higher amounts by their granulosa luteal cells. Vascular endothelial growth factor was accumulated in lesser concentrations in the FF of patients with endometriosis. Interleukin-1beta levels did not show significant changes. Implantation rates were decreased significantly in patients with endometriosis who were undergoing IVF. CONCLUSION(S) The data demonstrate that cytokines are regulated differently in patients with endometriosis, who have increased IL-6 production, and suggest that fine hormonal modulation of this cytokine occurs at the systemic and local (ovarian) levels. These changes show that the endocrine, paracrine, and autocrine milieu is different in patients with endometriosis and may be related to their lower implantation rates.

The pathogenesis of ovarian hyperstimulation syndrome: in vivo studies investigating the role of interleukin-1β, interleukin-6, and vascular endothelial growth factor

OBJECTIVE To evaluate systemic and ovarian changes in levels of interleukin (IL)-1beta, IL-6, and vascular endothelial growth factor (VEGF) in response to hCG administration to determine which may be the potential initiator of vascular effects and to identify the main source of the substance; to evaluate serum and follicular fluid levels of these cytokines as markers of ovarian hyperstimulation syndrome (OHSS), and to compare levels of these cytokines under basal conditions in women with normal ovulation and those with polycystic ovary syndrome (PCOS). DESIGN Prospective controlled study. SETTING In vitro fertilization program at the Instituto Valenciano de Infertilidad, Valencia, Spain. PATIENT(S) Women undergoing IVF, in whom the first two study objectives were analyzed, and women with normal ovulation and patients with PCOS undergoing retrieval of immature oocytes in natural cycles or cycles stimulated for IUI but cancelled during induction of ovulation, in whom the third study objective was analyzed. INTERVENTION(S) Serum was collected before and after hCG administration, and follicular fluid was collected at ovum pick-up. MAIN OUTCOME MEASURE(S) Serum and follicular fluid levels of IL-1beta, IL-6, and VEGF. RESULT(S) There was a significant increase in serum VEGF levels after hCG administration in patients who were at risk for OHSS compared with those who were not at risk for OHSS. Significantly lower VEGF levels were found in the follicular fluid of patients who were at risk; this decrease was the only useful marker to discriminate between the two groups. Moreover, both groups had similar cytokine production under basal conditions. An increase in serum E2 occurred coincident with a decrease in IL-1beta, IL-6, and VEGF in patients with PCOS. CONCLUSION(S) Vascular endothelial growth factor seems to be the mediator of hCG on the vascular tree. There was an early systemic increase in VEGF that may have significance in the development of OHSS. A decrease in the follicular fluid VEGF concentration is a valid marker to identify women in whom OHSS will develop. The pattern of cytokine release in patients with PCOS under basal conditions was not different from that in women with normal ovulation.

Evaluation of the ovarian reserve in young low responders with normal basal levels of follicle-stimulating hormone using three-dimensional ultrasonography

OBJECTIVE To assess the ovarian content of selectable (2-5 mm) follicles using three-dimensional ultrasonography in low responders to ovarian stimulation for IVF. DESIGN Prospective case-control study. SETTING IVF program at the Instituto Valenciano de Infertilidad. PATIENT(S) Ten low responders < or =35 years of age with normal basal serum FSH and eight control patients with normal response in a previous cycle. INTERVENTION(S) Blood was drawn under basal (day 3) conditions. Three-dimensional ultrasound was performed in both ovaries using a vaginal probe. MAIN OUTCOME MEASURE(S) Basal serum E2 and FSH measurements. The ovarian volume and the number of follicles > or =2 mm in each ovary were recorded and compared between the groups. RESULT(S) Low-responder women had significantly higher serum FSH levels than controls despite having FSH values within the normal range. The number of selectable follicles and the total number of follicles with an antrum were significantly decreased in low responders as compared with normal responders. Ovarian volume did not differ between the groups. CONCLUSION(S) This study introduces three-dimensional ultrasound as a novel method for the evaluation of low responders. The results show that the most plausible explanation for low response in young women with normal serum FSH is diminished ovarian reserve.

The Role of Gonadotropin-Releasing Hormone in Murine Preimplantation Embryonic Development

Previous studies have established the presence of an extrahypothalamic GnRH in a variety of tissues. GnRH receptor is known to be present in the placenta, which produces and secretes the decapeptide from the very early stages of placentation. We hypothesized that GnRH may play a role in the preimplantation development of embryos. To examine this hypothesis, we assessed GnRH and GnRH receptor messenger RNA (mRNA; RT-PCR) and protein expression (Immunohistochemistry) in preimplantation murine embryos at various developmental stages. Furthermore, preimplantation murine embryos were cultured with GnRH agonist and antagonist in vitro to assess the influence of GnRH analogs on embryo development. GnRH is expressed in the developing mouse embryo from morula to hatching blastocyst stages at the mRNA and protein levels. GnRH receptor mRNA is also present in the developing embryos studied. Preimplantation embryonic development was significantly enhanced by incubation with increasing concentrations of GnRH agonist a...

In vitro fertilization as a diagnostic and therapeutic tool in a patient with partial 17,20-desmolase deficiency

OBJECTIVE To present a case with 17,20-desmolase activity deficiency in which in vitro fertilization (IVF) served not only as a therapeutic approach but also as a diagnostic tool for the specificity of the enzymatic deficiency. DESIGN IVF in the patient under study compared with a control group. All women treated with pure follicle-stimulating hormone (FSH). SETTING IVF program at the Instituto Valenciano de Infertilidad. PATIENTS, PARTICIPANTS A patient with primary amenorrhea, who was the subject under study, and seven normally cycling control patients undergoing IVF in the same series. INTERVENTIONS IVF, steroidogenesis in vitro of granulosa-luteal cell obtained at ovum pick-up. MAIN OUTCOME MEASURE(S) Oocyte fertilization and embryo cleavage. Serum and follicular fluid (FF) levels of estradiol (E2), progesterone (P), testosterone (T), androstendione (A), 17 alpha-hydroxyprogesterone (17-OHP). In vitro accumulation of E2 and P. RESULTS Ovulation induction with FSH was successful in achieving follicular development despite low circulating E2. Fertilization and cleavage rates were similar to the control subjects. The patient developed ovarian hyperstimulation. The lack of 17,20-desmolase activity was detected by normal P levels in serum and FF, high 17-OHP, and low T, A, and E2 levels in serum and FF. Granulosaluteal cell cultures in the presence of T restored normal E2 and P production in response to gonadotropins. CONCLUSIONS In patients with 17,20-desmolase deficiency, follicular development, oocyte maturation, and fertilization can take place in a low estrogenic environment.

The follicular endocrine environment in stimulated cycles of women with endometriosis: steroid levels and embryo quality

OBJECTIVE To assess the endocrine milieu in follicles of stimulated cycles comparing women with and without endometriosis. Steroids were measured in follicular fluid (FF) and in in vitro culture of granulosa-luteal cells, and this status was related to the quality of the embryos obtained after IVF. DESIGN Case-control study. SETTING IVF program at the Instituto Valenciano de Infertilidad. PATIENT(S) Twenty-four women with laparoscopically documented endometriosis and 26 controls undergoing IVF. INTERVENTION(S) Individual follicular aspiration, oocyte isolation, FF storage, and preparation of luteinized granulosa cells for culture; oocyte insemination and embryo cleavage in standard IVF. MAIN OUTCOME MEASURE(S) Serum (day of ovum pickup) and FF measurements of estradiol, progesterone, testosterone, and androstenedione. Secretion of progesterone was measured in the cell-conditioned medium. Results were compared between patients with endometriosis and controls, as well as between oocytes that yielded embryos of different quality. RESULT(S) Levels of progesterone in the FF increased with the severity of the disease, whereas testosterone accumulation in the FF decreased with the severity of the disease. An increase in progesterone accumulation in vitro was observed in basal and hCG-induced granulosa cell cultures. No difference was observed in terms of embryo quality, and no steroid marker was able to identify follicles with oocytes that displayed embryos of good or bad quality under the inverted microscope. CONCLUSION(S) The data show differences in the steroidogenesis of follicles from stimulated women with and without endometriosis. These changes indicate good endocrine health but are not predictive of embryo quality.

Use of three-dimensional ultrasonography for the study of normal and pathologic morphology of the human embryo and fetus: preliminary report.

Our objective was to determine whether three‐dimensional ultrasonography offers advantages over two‐dimensional sonography for the evaluation of normal and pathologic morphology of human embryos and fetuses at various stages of pregnancy. Our studies suggest that small fetal and embryonic malformations are better defined with 3D sonography. Our experience indicates that 3D sonography allows more detailed visualization of fetal internal structures. We believe that with technical improvements 3D sonography will permit a more complete evaluation of fetuses earlier in gestation than is possible with current 2D sonographic instruments.

Cycles triggered with GnRH agonist: exploring low-dose HCG for luteal support

The aim of this study in patients at risk of ovarian hyperstimulation syndrome (OHSS) was to determine the efficacy and safety of luteal support using human chorionic gonadotrophin (HCG) after triggering ovulation with gonadotrophin-releasing hormone (GnRH) agonist in IVF/intracytoplasmic sperm injection antagonist cycles. A total of 192 OHSS-risk patients, following a GnRH antagonist protocol (0.25mg/day cetrorelix) during recombinant FSH stimulation, were triggered with 1.5mg s.c. leuproreline for ovulation. A total of three boluses of HCG were used for luteal support, 1000IU (group A, n=44), 500IU (group B, n=115) or 250IU (group C, n=33) every third day, starting the day after oocyte retrieval. For the reproductive outcome, main variables were biochemical and clinical pregnancy rates, and for OHSS, the variables were the numbers of moderate and severe OHSS cases. Overall pregnancy rate was 51.8% and clinical pregnancy rate was 43.4%. This study observed eight cases of moderate (4.2%) and seven of severe OHSS (3.6%). Six out of the seven (85.7%) severe cases were late-onset OHSS, related to pregnancy. In conclusion, GnRH agonist single dose for triggering ovulation and low doses of HCG used as luteal-phase support seem to secure a normal pregnancy outcome without increasing the OHSS risk.

Reducing blood loss at myomectomy with use of a gelatin-thrombin matrix hemostatic sealant

OBJECTIVE To evaluate the hemostatic efficacy and handling of gelatin-thrombin matrix in abdominal myomectomy. DESIGN Prospective and randomized trial. SETTING University teaching hospital. PATIENT(S) Women (n = 50) with uterine fibroids with a uterine size equivalent to > or =16 weeks gestation. INTERVENTION(S) Gelatin-thrombin matrix (FloSeal Matrix; Baxter Healthcare Corp., Fremont, CA) was delivered to the site of the uterine bleeding during myomectomy. MAIN OUTCOME MEASURE(S) Patient age, parity, number of myomas, operative time, blood loss, transfusion, intraoperative and postoperative complications, and length of hospitalization were evaluated. RESULT(S) The average blood loss during surgery was 80 +/- 25.5 mL for the FloSeal group and 625 +/- 120.5 mL for the control group. Intraoperative blood transfusion was necessary in five patients from the control group. Postoperative blood loss was 25 +/- 5 mL for the FloSeal group and 250 +/- 75 mL for the control group. Length of the postoperative hospital stay was 2.5 +/- 1.2 days for FloSeal group and 4.5 +/- 1.3 for the control group. No major immediate or delayed complications were observed in either group. CONCLUSION(S) Reductions in hemorrhage in FloSeal-treated women undergoing a myomectomy are encouraging, and provide evidence for the ability of gelatin-thrombin matrix to reduce blood loss when applied immediately and directly to bleeding uterine tissue.

Polycystic ovarian syndrome: assessment with color Doppler angiography and three-dimensional ultrasonography.

We conducted a three‐dimensional ultrasonographic evaluation of the size and structure of the ovaries of women who had clinical and biochemical findings suggestive of polycystic ovary syndrome. We carried out a comparative color Doppler frequency and color Doppler amplitude study of the vascular patterns of these ovaries. This study involved 65 women of reproductive age with polycystic ovary syndrome and 25 eumenorrheic women who were not taking hormonal contraceptives and who had a body mass index below 25 kg/m2. Compared to controls, women with polycystic ovary syndrome had larger ovaries and thicker stroma, increased impedance in the uterine arteries, increased stromal vascularity with decreased impedance that persisted throughout the menstrual cycle, and a lack of luteal conversion.