Francisco Raga

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...

Autologous cell therapy with CD133+ bone marrow-derived stem cells for refractory Asherman's syndrome and endometrial atrophy: a pilot cohort study

STUDY QUESTION Could cell therapy using autologous peripheral blood CD133+ bone marrow-derived stem cells (BMDSCs) offer a safe and efficient therapeutic approach for patients with refractory Ashermans syndrome (AS) and/or endometrial atrophy (EA) and a wish to conceive? SUMMARY ANSWER In the first 3 months, autologous cell therapy, using CD133+ BMDSCs in conjunction with hormonal replacement therapy, increased the volume and duration of menses as well as the thickness and angiogenesis processes of the endometrium while decreasing intrauterine adhesion scores. WHAT IS KNOWN ALREADY AS is characterized by the presence of intrauterine adhesions and EA prevents the endometrium from growing thicker than 5 mm, resulting in menstruation disorders and infertility. Many therapies have been attempted for these conditions, but none have proved effective. STUDY DESIGN, SIZE, DURATION This was a prospective, experimental, non-controlled study. There were 18 patients aged 30-45 years with refractory AS or EA were recruited, and 16 of these completed the study. Medical history, physical examination, endometrial thickness, intrauterine adhesion score and neoangiogenesis were assessed before and 3 and 6 months after cell therapy. PARTICIPANTS/MATERIALS, SETTING, METHODS After the initial hysteroscopic diagnosis, BMDSC mobilization was performed by granulocyte-CSF injection, then CD133+ cells were isolated through peripheral blood aphaeresis to obtain a mean of 124.39 million cells (range 42-236), which were immediately delivered into the spiral arterioles by catheterization. Subsequently, endometrial treatment after stem cell therapy was assessed in terms of restoration of menses, endometrial thickness (by vaginal ultrasound), adhesion score (by hysteroscopy), neoangiogenesis and ongoing pregnancy rate. The study was conducted at Hospital Clínico Universitario of Valencia and IVI Valencia (Spain). MAIN RESULTS AND THE ROLE OF CHANCE All 11 AS patients exhibited an improved uterine cavity 2 months after stem cell therapy. Endometrial thickness increased from an average of 4.3 mm (range 2.7-5) to 6.7 mm (range 3.1-12) ( ITALIC! P = 0.004). Similarly, four of the five EA patients experienced an improved endometrial cavity, and endometrial thickness increased from 4.2 mm (range 2.7-5) to 5.7 mm (range 5-12) ( ITALIC! P = 0.03). The beneficial effects of the cell therapy increased the mature vessel density and the duration and intensity of menses in the first 3 months, with a return to the initial levels 6 months after the treatment. Three patients became pregnant spontaneously, resulting in one baby boy born, one ongoing pregnancy and a miscarriage. Furthermore, seven pregnancies were obtained after fourteen embryo transfers, resulting in three biochemical pregnancies, one miscarriage, one ectopic pregnancy, one baby born and one ongoing pregnancy. LIMITATIONS, REASONS FOR CAUTION Limitations of this pilot study include the small sample size and the lack of control group. WIDER IMPLICATIONS OF THE FINDINGS This novel autologous cell therapy is a promising therapeutic option for patients with these incurable pathologies and a wish to conceive. STUDY FUNDING/COMPETING INTERESTS This study was funded by the Spanish Ministry of Science and Innovation (SAF 2012-31017, Principal Investigator C.S.), Spanish Ministry of Health (EC11-299, Principal Investigator C.S.) and Regional Valencian Ministry of Education (PROMETEOII/2013/018, Principal Investigator C.S.). Four authors (X.S., I.C., A.P. and C.S.) are co-inventors of the patent resulting from this work (Application number: 62/013,121). S.C., C.A., F.R., J.F., J.P. and J.R. have no conflict of interest in relation to this work. TRIAL REGISTRATION NUMBER This study was registered with ClinicalTrials.gov (NCT02144987).

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.

Fetal megacystis: differential diagnosis.

The purpose of our retrospective observational series was to determine whether the sonographic characteristics of fetal megacystic bladders can be used to reliably establish the most likely diagnosis in fetuses with this condition. The sonographic records of pregnant patients referred to our institutions over a 10‐year period who were found on initial 2‐dimensional sonography to be carrying fetuses with megacystis were examined for evidence of a keyhole sign, bladder thickness, amniotic fluid index, and fetal sex. When available, 3‐/4‐dimensional sonography, Doppler angiography, tomographic ultrasound imaging, virtual organ computer‐aided analysis, and automatic volume calculation were used as part of the detailed fetal anatomic survey. Twenty fetuses with megacystis were identified. Seventeen were male; 2 were female; and 1 had ambiguous genitalia. All male fetuses with megacystis originally had a diagnosis of prune belly syndrome. The diagnosis for 10 male fetuses with a keyhole sign was changed to megacystis secondary to posterior urethral valves. The fetus with ambiguous genitalia had prune belly syndrome. One of the female fetuses had a diagnosis of urethral atresia, and the diagnosis for the other female fetus was megacystis‐microcolon‐intestinal hypoperistalsis syndrome. In conclusion, in fetuses with megacystic bladders, it is possible to distinguish between cases with prune belly syndrome, posterior urethral valves, urethral atresia, and megacystis‐microcolon‐intestinal hypoperistalsis syndrome by a detailed anatomic survey using 2‐ and 3‐/4‐dimensioinal sonographic techniques.

Is avoidance of surgery possible in patients with perimenopausal ovarian tumors using transvaginal ultrasound and duplex color Doppler sonography

A total of 108 cases of small perimenopausal ovarian tumors (largest diameter, less than 7 cm) were evaluated by transvaginal sonography and duplex color Doppler sonography; 101 had criteria for benignity and 7 for malignancy. All cysts were punctured and aspirated to avoid unnecessary surgery (benign cases) and establish the cytologic diagnosis as well as to avoid the risk of leakage (malignant cases). Recurrence rate of benign punctured cysts was 25% within 1 year of follow up. No difference in recurrent rate was seen between pre‐ and postmenopausal patients (19 of 56 cases in the premenopausal group, and five of 28 cases in the postmenopausal group). The larger the cyst, the greater the risk of recurrence. Puncture and aspiration of seven sonographically established malignant tumors allowed cytologic diagnosis before surgery, and no evidence of leakage was noted at the time of surgery. No significant differences were observed between cytologic and histopathologic findings in cases that went to surgery. Puncture and aspiration of small endometriomas (17 cases) was found to be inefficient for therapeutic purposes. Finally, owing to the high percentage of unsatisfactory cytologic results (20%) with cyst aspiration, vaginal and color Doppler sonography seemed more efficient in distinguishing between benignity and malignancy.

Immunoreactive gonadotropin-releasing hormone expression in cycling human endometrium of fertile patients.

OBJECTIVE To investigate the protein expression of GnRH in the endometrium of fertile patients throughout the menstrual cycle. DESIGN Prospective longitudinal study. SETTING Department of Gynecology and Obstetrics, Reproductive Immunology Laboratory, Stanford University Medical Center. PATIENT(S) Twenty-two fertile premenopausal women submitted to laparoscopic surgery for benign gynecologic indications. None of the 22 women had endometriosis or pelvic inflammatory disease. INTERVENTION(S) An endometrial biopsy specimen using the Novak curette was obtained at the time of surgery. MAIN OUTCOME MEASURE(S) Protein expression and localization from unfractioned endometrial tissue was analyzed by immunohistochemistry. RESULT(S) Gonadotropin-releasing hormone is expressed at the protein level in both the endometrial stroma and epithelium throughout the entire menstrual cycle of fertile women. Immunostaining in the human epithelium reached maximal levels in the midluteal phase and was elevated in the stroma throughout the entire luteal phase. CONCLUSION(S) Our results demonstrate the presence of GnRH in the human endometrium at the protein level throughout the entire menstrual cycle of fertile women, with an increase in the luteal phase compared with the preovulatory endometrium.

First-trimester neck abnormalities: three-dimensional evaluation.

In order to study the first trimester ultrasonographic differences between nuchal translucency and hygroma colli, we rescanned 25 fetuses (13 with nuchal translucency and 12 with hygroma colli) using transvaginal and three‐dimensional ultrasonography, after obtaining a fetal karyotype report. Our objective was to test the premise that the different physiopathologic mechanisms of both processes would be reflected in detectable sonographic differences. Our retrospective analysis showed that the most striking ultrasonographic difference was the presence of bullae as well as greater irregularity, extent, and amplitude of the membrane in cases of hygroma colli. Fetuses with simple nuchal translucency had a more homogeneous linear membrane. Although detailed analysis was impossible in 30% of cases, we found three‐dimensional ultrasonography to be a useful technique for establishing the differences between these two entities.

Early detection of embryonic malformations by transvaginal and color Doppler sonography.

A total of 834 women with uneventful pregnancies were followed prospectively until the 15th week of gestation by TVS to screen for early embryonic malformations. Twenty‐eight embryonic anomalies were detected in this series (3.3%). The median gestational age at diagnosis was 11 (range, 8 to 15) weeks. Two neural tube defects were missed by early TVS screening. Two suspected abdominal wall defects were not confirmed by repeat mid‐second trimester abdominal sonography representing 6.7% of all fetal malformations evident by 24 weeks of gestation. The sensitivity and specificity of TVS screening for fetal malformations in this series were 93.3 and 99.7%, respectively. In addition, the role of TCDU also was investigated in these 28 abnormal embryos. TCDU proved to be of limited value as it was useful only for evaluating those embryonic malformations with vascular involvement. In such cases, the diagnosis was greatly enhanced with this technique. Two previously undetected single umbilical arteries were also diagnosed by TCDU as well. This series included 11 aneuploid embryos (44%), most of them among the embryos with focal or generalized edema. This study demonstrates that screening for early embryonic malformations is feasible using TVS techniques, but a repeat mid‐second trimester abdominal sonogram still is recommended.

Cotyledonoid dissecting leiomyoma of the uterus

We describe a cotyledonoid dissecting leiomyoma in a 33-year-old nulliparous woman treated by laparoscopy.

Fetus acardius: two- and three-dimensional ultrasonographic diagnoses.

To determine whether ultrasonographic detection of acardiac fetuses and diagnostic accuracy of related malformations improve with complementary use of two‐dimensional ultrasonography, three‐dimensional ultrasonography, and Doppler scanning.