Ido Ben-Ami

In vitro maturation of human germinal vesicle-stage oocytes: role of epidermal growth factor-like growth factors in the culture medium

BACKGROUND In vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side effects of gonadotrophin stimulation for IVF. The pregnancy rates from oocytes matured in vitro are still lower than those of in vivo stimulation cycles, indicating that optimization of IVM remains a challenge. Recently, it was demonstrated that LH exerts its action on ovulation, at least in part, through stimulation of the production of the epidermal growth factor family members amphiregulin (Areg) and epiregulin (Ereg) in pre-ovulatory follicles, and they, in turn, serve as paracrine mediators of LH. We aimed to investigate the effect of supplementation of the medium with Areg and Ereg on the maturation rate of immature oocytes. METHODS A total of 105 sibling human germinal vesicle (GV) oocytes obtained after gonadotrophin stimulation were cultured in a complex defined medium either with or without supplemented recombinant human Areg (75 ng/ml) and Ereg (75 ng/ml) for 24 h. RESULTS Significantly more oocytes reached the metaphase II stage at 24 h in media supplemented with Areg and Ereg (75.5 versus 36.5%, P < 0.001). In vitro matured oocytes retrieved from the two subgroups had no statistically significant difference in fertilization and cleavage rates or morphology scores. Overall, a significantly higher number of Day 2 (52.8 versus 26.9% P < 0.01) and Day 3 (45.2 versus 23%, P < 0.05) embryos originated from GV oocytes cultured in the Areg- and Ereg-enriched medium. CONCLUSIONS Supplementation of the maturation medium with Areg and Ereg improves the maturation of human GV oocytes in vitro.

The Major Histopathologic Characteristics in the Vulvar Vestibulitis Syndrome

Objective: In order to better understand the etiology of the vulvar vestibulitis syndrome, we examined the histopathologic parameters in vestibular mucosa, and compared the findings in specimens obtained from women with vulvar vestibulitis with those obtained from the control group. Study Design: Specimens of vestibulitis were obtained from 24 patients, undergoing circumferential vestibulectomy under general anesthesia due to the symptoms and signs consistent with vulvar vestibulitis. Control specimens were obtained from 16 women, without symptoms or signs of vulvar vestibulitis, undergoing reconstructive introital surgery due to roomy vagina, rectocele or painful episiotomy. All vestibular tissue specimens were examined for intensity of inflammation, extension of inflammatory cells into the epithelium, vascular proliferation, the presence of mast cells and proliferation of peripheral nerve bundles. Results: No significant difference was found regarding the degree of inflammation, the extension of inflammatory cells into the epithelium, the vascular proliferation and the presence of mast cells while comparing the study and the control groups. The only histopathologic feature, differentiating the patients with vulvar vestibulitis from the control group, was the proliferation of peripheral nerve bundles found in 19 out of 24 (79.1%) specimens expressing vestibulitis and in none of 16 control specimens (p < 0.0001). Conclusion: Our results, therefore, support the existence of peripheral nerve hyperplasia in vestibular tissue obtained from patients with vulvar vestibulitis, and exclude the role of active inflammation or mast cells as probable etiologies for the vulvar vestibulitis syndrome.

Intracytoplasmic sperm injection outcome of ejaculated versus extracted testicular spermatozoa in cryptozoospermic men

OBJECTIVE To compare intracytoplasmic sperm injection (ICSI) outcome of patients with cryptozoospermia after use of ejaculated versus testicular sperm in different cycles of the same patients. DESIGN Retrospective cohort study. SETTING University-affiliated infertility center. PATIENT(S) A total of 17 patients with cryptozoospermia who underwent a total of 116 ICSI cycles. INTERVENTION(S) The patients initially underwent several ICSI cycles using ejaculated sperm (n = 68, 58.6%) that were followed by ICSI cycles using testicular sperm (n = 48, 41.4%). MAIN OUTCOME MEASURE(S) Fertilization rate, pregnancy rate (PR). RESULT(S) There were no significant differences in fertilization rates between the two subgroups. A comparison between testicular sperm extraction (TESE) versus ejaculated sperm cycles revealed significantly higher implantation rate (20.7% vs. 5.7%), higher PR (42.5% vs. 15.1%), and higher take home baby rate (27.5% vs. 9.4%). A multivariable logistic regression analysis showed three significant predictors for pregnancy, namely the use of testicular sperm (odds ratio [OR] 5.1, 95% confidence interval [95% CI] 1.8-14.8), use of motile sperm (OR 12.9, 95% CI 2.1-79.1), and female age (OR 0.83, 95% CI 0.7-0.9). CONCLUSION(S) Testicular sperm extraction is justified in patients with cryptozoospermia who fail to conceive by ICSI using ejaculated spermatozoa, as it offers higher PR.

EGF-like growth factors as LH mediators in the human corpus luteum

BACKGROUND This study aims to investigate the role of epidermal growth factor-like ligands, amphiregulin (Ar) and epiregulin (Ep), in regulation of apoptosis in luteinized human granulosa cells. METHODS Luteinized human granulosa cells were obtained from women undergoing IVF treatment. Ar and Ep mRNA levels were measured by real-time RT-PCR. The rate of apoptosis was measured by TUNEL. Progesterone levels were measured using radioimmunoassay. Ar- and Ep-induced activation of signaling cascades and Ar protein levels were detected by western blotting. RESULTS LH stimulation of luteinized human granulosa cells induced biosynthesis of Ar and Ep mRNA in a time-dependent manner. The blockade of MEK (by U0126) reduced the expression of LH-induced Ar and Ep biosynthesis. Incubation of the cells with Ar and Ep completely abolished the increase in apoptosis rate induced by serum starvation, and concomitantly caused a pronounced increase in progesterone production. Stimulation of the cells with Ar and Ep also activated the ERK and AKT signaling cascades. Finally, we demonstrated that the pro-survival effect of Ar and Ep is partially dependent on their ability to induce progesterone production. CONCLUSIONS Ar and Ep serve as pro-survival LH mediators in the human corpus luteum.

Does intraoperative spillage of benign ovarian mucinous cystadenoma increase its recurrence rate

OBJECTIVE To report a higher than estimated recurrence rate of benign mucinous cystadenomas after complete resection of the first one, and to assess potential risk factors for recurrence after complete surgical excision. STUDY DESIGN We retrospectively reviewed all cases of women who underwent either laparoscopic or laparotomic removal of benign mucinous adnexal cysts by either adnexectomy or cystectomy in our institution between 1996 and 2006. RESULTS Included were the data of 42 women who fulfilled study entry criteria. Three of them (7.1%) underwent a second operation because of a recurrence of the lesion. A significantly higher rate of women who had cyst recurrence had undergone cystectomy as opposed to adnexectomy (P < .05). Intraoperative rupture of cysts during cystectomy was also significantly associated with cyst recurrence (P < .03). CONCLUSION Mucinous cystadenoma recurrence is apparently not as rare as reported in the literature. Intraoperative cyst rupture and cystectomy instead of adnexectomy emerged as being two risk factors for recurrence.

The Role of Pigment Epithelium-Derived Factor in the Pathophysiology and Treatment of Ovarian Hyperstimulation Syndrome in Mice

CONTEXT Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of assisted reproduction. OHSS is induced by an ovarian release of vasoactive, angiogenic substances that results in vascular hyperpermeability, leakage, and shift of fluids from blood vessels into the extravascular space with consequent ascites and edema that are attributed to vascular endothelial growth factor (VEGF). OBJECTIVE Our objective was to examine a physiological approach for preventing and treating OHSS, based on negating the VEGF network. DESIGN We used a mouse OHSS model and cultured granulosa cells. MAIN OUTCOME Changes in pigment epithelium-derived factor (PEDF) and VEGF were measured by quantitative PCR and Western blot analysis. OHSS was recorded by changes in body weight and in peritoneal vascular leakage, quantified by the modified Miles vascular permeability assay. RESULTS Granulosa cells produced and secreted the anti-angiogenic factor, PEDF, in an inverse fashion to VEGF. The physiological PEDF-VEGF counterbalance was found to be impaired in the mouse OHSS model. Treatment of OHSS-induced mice with low doses of recombinant PEDF (rPEDF) alleviated OHSS signs including edema (P < .001) and vascular leakage (P < .001) and reduced the level of ovarian VEGF mRNA. Low doses of rPEDF also reduced VEGF mRNA levels in granulosa cells in vitro. However, these effects were not seen at higher doses of rPEDF, suggesting a hormetic mechanism of rPEDF action. CONCLUSION These observations provide a new perspective into the pathophysiology of OHSS, namely, high expression level of VEGF together with a nearly undetectable level of PEDF. A replacement therapy with rPEDF is suggested as an innovative physiological treatment for OHSS. Finally, control of the PEDF-VEGF reciprocal relationship could open new therapeutic avenues for other angiogenic-related fertility pathologies.

Hysteroscopic management of retained products of conception: meta-analysis and literature review

OBJECTIVE Hysteroscopic removal of retained products of conception (RPOC) may allow complete removal of RPOC and decreased rates of intrauterine adhesions (IUA) when compared to the traditional blind curettage. The aim of this meta-analysis is to examine the current evidence regarding the use of hysteroscopy for treatment of RPOC. STUDY DESIGN A literature search was conducted in December 2012 using MEDLINE and ClinicalTrials. The study selection criteria were use of the standard hysteroscopic technique for removal of RPOC in 5 or more cases, in any study design. We reviewed 11 studies, of which 5 retrospective studies met the selection criteria (comprising 326 cases). The rates of incomplete RPOC removal, surgical complications, post-operative IUA and subsequent pregnancies were abstracted and weighted events rates using a fixed meta-analysis model were calculated. RESULTS Only one study compared the rates of IUA following hysteroscopy and curettage, precluding a meta-analysis comparison of the two techniques. There were no cases of incomplete RPOC removal. Three perioperative complications occurred (uterine perforation, infection, and vaginal bleeding). IUA on follow-up hysteroscopy were found in 4/96 women (weighted rate of 5.7%, 95% CI 2.4%, 13.0%). Of the 120 women desiring a subsequent pregnancy 91 conceived (weighted rate of 75.3%, 95% CI 66.7%, 82.3%). CONCLUSIONS The lack of traditional curettage comparison groups in most studies precludes the conclusion that hysteroscopy is superior to traditional curettage, but this procedure does appear to have low complication rates, low rates of IUA, and high rates of subsequent pregnancies.

Prenatal diagnosis of sex chromosome abnormalities: the 8-year experience of a single medical center.

Objective: To assess the indications for prenatal karyotyping of sex chromosomal abnormalities (SCAs) during pregnancy. Methods: All singleton pregnancies interrupted in our institute because of SCAs (1998–2005) were categorized into subgroups of 45,XO (Turner syndrome), 47,XXY (Klinefelter syndrome), 47,XXX and 47,XYY. The indications for prenatal diagnostic testing were recorded. Results: There were 67 SCAs pregnancies: 33% Turner syndrome, 28% Klinefelter syndrome, 21% 47,XXX and 18% 47,XYY. Maternal age was similar among the 4 groups (34 ± 5, range 25–42 years). The main indications for fetal karyotyping were abnormal Down’s syndrome (DS) screening or ultrasound findings, advanced maternal age (≧35 years), and parental request. About 2/3 of the Turner and 47,XYY cases had either abnormal DS screening tests or sonographic findings, such as: increased nuchal translucency, mainly cystic hygroma and fetal hydrops. However, fetal karyotyping in more than 2/3 of the 47,XXX and 47,XXY cases was mainly performed because of advanced maternal age, and the diagnosis of fetal SCAs was coincidental (p <0.03). Conclusions: Our recent suggestion to expand the DS screening capacity to other chromosomal abnormalities including SCAs is further supported. Prenatal detection seems to be promising for Turner syndrome and possibly for 47,XYY syndrome, while other SCAs are less likely to be detected either by ultrasound or biochemical screening.

Do assisted-reproduction twin pregnancies require additional antenatal care?

Iatrogenic twinning has become the main side-effect assisted reproduction treatment. We have evaluated the evidence for additional care that assisted-reproduction twins may require compared with spontaneous twins. Misacarriages are increased in women with tubal problems and after specific treatments. Assisted-reproduction twin pregnancies complicated by a vanishing twin after 8 weeks have an increased risk of preterm delivery and of low and very low birthweight compared with singleton assisted-reproduction pregnancies. Monozygotic twin pregnancies occur at a higher rate after assisted reproduction treatment and are associated with a higher risk of perinatal complications. The incidence of placenta praevia and vasa praevia is increased in assisted-reproduction twin pregnancies. Large cohort studies do not indicate a higher rate of fetal congenital malformations in assisted-reproduction twins. Overall, assisted-reproduction twins in healthy women <45 years of age are not associated with a notable increase in antenatal complication rates and thus do not require additional antenatal care compared with spontaneous twins. The risks of maternal and fetal morbidity and mortality associated with assisted-reproduction twins is only increased in women with a pre-existing medical condition such as hypertensive disorders and diabetes and most of these risks can be avoided with single-embryo transfer. Following the birth of the first IVF baby, rumours started to spread in both the medical literature and the media about the long-term health effects for children born following assisted reproduction treatment. However, after more than 30 years, the most common complications associated with IVF treatment remain indirect and technical such as the failure of treatment and ovarian hyperstimulation. Iatrogenic twinning has become the main side-effect of assisted reproduction treatment and the increasing number of twin pregnancies, in particular in older women, has generated numerous debates on the need for additional healthcare provision. In this review, we have evaluated the evidence for additional care that assisted-conception twin pregnancies may require compared with spontaneous twin pregnancies. Twin pregnancies are obviously at higher risk of perinatal complications than singletons due to a natural increase in the incidence of fetal anomalies, antenatal disorders and obstetric and neonatal complications associated with the development of two fetuses instead of one. Overall, our review indicates that some antenatal complications are more frequent in assisted-conception twin pregnancies than in spontaneous twin pregnancies but their prevalence is low and thus their impact on the morbidity and mortality of an individual assisted-conception twin pregnancy is limited. Assisted reproduction treatment has become available to older women with pre-existing maternal medical conditions such as chronic hypertension and diabetes. The increased obstetrical risks in this population must be considered prior to attempts at assisted conception, and the transfer of more than one embryo should be avoided in women with a pre-existing maternal medical condition.

Termination of Pregnancy due to Fetal Abnormalities Performed after 23 Weeks’ Gestation: Analysis of Indications in 144 Cases from a Single Medical Center

Background/Aims: To assess the indications for late termination (≥23 weeks’ gestation) of pregnancy (LTOP), and to evaluate the rate of cases potentially diagnosable earlier. Methods: Cases of singleton pregnancy ending in LTOP due to fetal abnormalities in our institute between 1/1998 and 12/2005 were retrospectively reviewed. The women were divided into two groups according to the sequence of events that led to LTOP: Group 1 – the first test indicating an abnormal finding was performed ≤23 weeks’ gestation, but LTOP was performed >23 weeks; Group 2 – the first test indicating an abnormal finding was performed ≥23 weeks of gestation, or the fetal prognosis was not certain at the time of diagnosis and there was a medical recommendation to continue investigation. Results: There were 144 cases of LTOP (average gestational age 26.2 ± 3.4 weeks). More than 70% of the cases were aborted because of chromosomal/genetic indication in Group 1; many of them could have been detected earlier in pregnancy, while about 80% of the cases were aborted because of structural abnormalities in Group 2 (p < 0.001). The structural anomaly could have been diagnosed earlier in 56 cases (∼74%) if the pregnant woman had undergone an earlier anomaly scan. In another 13 cases (9%), fetal prognosis was not certain and continuing prenatal investigation was required. Conclusions: The most common indications for LTOP were structural abnormalities (91 cases, 70%) which included the central nervous system (26 cases, 29%), cardiac abnormalities (24 cases, 26%), and multiple malformations (18 cases, 20%). The diagnosis of fetal anomaly could have been made earlier in more than half of the pregnant women undergoing LTOP.