Alon Kedem

Prevascularization of cardiac patch on the omentum improves its therapeutic outcome

The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modality for regenerating the myocardium after myocardial infarction (MI). We present here a strategy for the engineering of a cardiac patch with mature vasculature by heterotopic transplantation onto the omentum. The patch was constructed by seeding neonatal cardiac cells with a mixture of prosurvival and angiogenic factors into an alginate scaffold capable of factor binding and sustained release. After 48 h in culture, the patch was vascularized for 7 days on the omentum, then explanted and transplanted onto infarcted rat hearts, 7 days after MI induction. When evaluated 28 days later, the vascularized cardiac patch showed structural and electrical integration into host myocardium. Moreover, the vascularized patch induced thicker scars, prevented further dilatation of the chamber and ventricular dysfunction. Thus, our study provides evidence that grafting prevascularized cardiac patch into infarct can improve cardiac function after MI.

The effect of sulfation of alginate hydrogels on the specific binding and controlled release of heparin-binding proteins

To mimic the high affinity of heparin-binding proteins to heparin/heparan sulfate, the uronic acids in non-sulfated alginate were sulfated, and hydrogels of mixed alginate/alginate-sulfate were fabricated. Surface plasmon resonance analysis probed the interactions of 13 proteins with alginate-sulfate. Of these, the 10 heparin-binding proteins revealed strong binding to alginate-sulfate and heparin, but not to alginate. The equilibrium binding constants to alginate-sulfate were comparable or one order of magnitude higher than those obtained between the proteins and heparin. Only the fibroblast growth factors (FGFs) revealed higher affinity for heparin than to alginate-sulfate. Sulfation of hyaluronan, as well, resulted in strong binding of basic FGF to hyaluronan-sulfate, but not to hyaluronan. Mixed hydrogels of alginate/alginate-sulfate sustained the release of basic FGF, with the release rate being dependent on the percentage of bFGF bound to the hydrogels. In vivo, the delivery of bFGF bound to alginate/alginate-sulfate scaffolds induced the formation of twice the number of blood vessels compared to when bFGF was delivered adsorbed to the matrix and 51% of the vessels were matured, as judged by pericyte coverage of the vessels. Our results thus describe the engineering of alginate hydrogels for the spatially presentation and controlled delivery of heparin-binding proteins.

Growth differentiating factor 9 (GDF9) and bone morphogenetic protein 15 both activate development of human primordial follicles in vitro, with seemingly more beneficial effects of GDF9.

CONTEXT The signals initiating growth of primordial follicles are unknown. Bone morphogenetic protein 15 (BMP15) and growth differentiating factor 9 (GDF9) are promising candidates. OBJECTIVE The objective of the study was to evaluate for the first time the effects of human recombinant BMP15 and human recombinant GDF9 on the in vitro development of human primordial follicles. DESIGN AND SETTING This was a controlled culture study performed in a major tertiary university-affiliated medical center. MATERIALS Materials included ovarian tissue from 17 girls/women and three aborted human fetuses. INTERVENTION There were no interventions. MAIN OUTCOME MEASURE Histological and immunohistochemical (proliferating cell nuclear antigen, BMP15, and GDF9) studies and an endocrine assay of 17β-estradiol were conducted. RESULTS In the samples from girls/women, the number of developing follicles was greater with GDF9 or BMP15 alone than with no BMP15 or GDF9. Higher 17β-estradiol secretion was noted after treatment with GDF9 than with BMP15 or with GDF9+anti-GDF9. The number of atretic follicles was greater with BMP15 than with GDF9. Proliferating cell nuclear antigen expression was greater with the higher dose of both growth factors than the lower dose. Expression of BMP15 and GDF9 was identified in samples cultured without BMP15 or GDF9. Results for the fetal follicles yielded no distinguishable pattern. CONCLUSIONS Although both BMP15 and GDF9 promoted activation of human primordial follicles from girls/women (but not human fetuses) in a dose-dependent manner, GDF9 seems more beneficial.

Does local injury to the endometrium before IVF cycle really affect treatment outcome? Results of a randomized placebo controlled trial

Aim: To evaluate the effect of local injury to the endometrium during spontaneous menstrual cycles before in vitro fertilization (IVF) treatment on implantation and pregnancy rates in women with recurrent implantation failure (RIF). Methods: In a prospective randomized controlled trial (RCT), a total of 36 patients, with RIF undergoing IVF, were randomized to two groups. In 18 patients, endometrial biopsies were performed using a pipelle curette on days 9–12 and 21–24 of the menstrual cycle preceding IVF treatment. In 18 control patients, a cervical pipelle was performed. Results: The implantation rate (2.08% versus 11.11%; p = 0.1), clinical (0% versus 31.25%; p < 0.05) and live births rates (0% versus 25%; p = 0.1) were lower in the experimental group compared with controls. Conclusion: Our RCT did not find any benefit from local injury to the endometrium in women with a high number of RIFs. Further studies are warranted to better define the target population of patients who may benefit from this procedure.

High expression of luteinizing hormone receptors messenger RNA by human cumulus granulosa cells is in correlation with decreased fertilization.

OBJECTIVE To elucidate the LH receptor (LHR) expression patterns in human granulosa cells (GCs) from antral to preovulatory stages, and to investigate a correlation to oocyte function. DESIGN Luteinized preovulatory GCs were obtained from preovulatory follicles aspirated during IVF (≥ 17 mm). The GCs from small- (<10 mm) and medium-sized (10-15 mm) follicles were obtained during in vitro maturation (IVM) procedures. Cumulus GCs were obtained during oocyte denudation for intracytoplasmatic sperm injection (ICSI) procedures (IVF). SETTING Referral center. PATIENT(S) Seventy IVF patients and 20 IVM patients. INTERVENTION(S) GC collection. MAIN OUTCOME MEASURE(S) The LHR expression levels in mural and cumulus GCs of different follicular sizes and their correlation to oocyte outcome. RESULT(S) The LHR expression increased with follicle size and was higher in mural GCs compared with cumulus cells. The LHR expression in cumulus GCs from preovulatory follicles was higher in metaphase II (MII) oocytes than in metaphase I or germinal vesicle oocytes (IVF). Unexpectedly, higher expression of LHR in cumulus GCs of MII oocytes correlated with decreased fertilization rates. CONCLUSION(S) The LHR expression in small follicles obtained in IVM suggests a role for hCG administration during IVM procedures. Overexpression of LHR in cumulus GCs of MII oocytes may signal malfunction of oocytes and low fertilization capacity.

Characterization of the human cumulus cell transcriptome during final follicular maturation and ovulation

Cumulus expansion and oocyte maturation are central processes in ovulation. Knowledge gained from rodent and other mammalian models has revealed some of the molecular pathways associated with these processes. However, the equivalent pathways in humans have not been thoroughly studied and remain unidentified. Compact cumulus cells (CCs) from germinal vesicle cumulus oocyte complexes (COCs) were obtained from patients undergoing in vitro maturation (IVM) procedures. Expanded CCs from metaphase 2 COC were obtained from patients undergoing IVF/ICSI. Global transcriptome profiles of the samples were obtained using state-of-the-art RNA sequencing techniques. We identified 1746 differentially expressed (DE) genes between compact and expanded CCs. Most of these genes were involved in cellular growth and proliferation, cellular movement, cell cycle, cell-to-cell signaling and interaction, extracellular matrix and steroidogenesis. Out of the DE genes, we found 89 long noncoding RNAs, of which 12 are encoded within introns of genes known to be involved in granulosa cell processes. This suggests that unique noncoding RNA transcripts may contribute to the regulation of cumulus expansion and oocyte maturation. Using global transcriptome sequencing, we were able to generate a library of genes regulated during cumulus expansion and oocyte maturation processes. Analysis of these genes allowed us to identify important new genes and noncoding RNAs potentially involved in COC maturation and cumulus expansion. These results may increase our understanding of the process of oocyte maturation and could ultimately improve the efficacy of IVM treatment.

Ongoing Pregnancy Rates in Women with Low and Extremely Low AMH Levels. A Multivariate Analysis of 769 Cycles

Background The ideal test for ovarian reserve should permit the identification of women who have no real chance of pregnancy with IVF treatments consequent upon an extremely reduced ovarian reserve. The aim of the current study was to evaluate pregnancy rates in patients with low AMH levels (0.2–1 ng/ml) and extremely low AMH levels (<0.2 ng/ml) and to determine the cumulative pregnancy rates following consecutive IVF treatments. Methods We conducted an historical cohort analysis at a tertiary medical center. Serum AMH levels were measured at initial clinic visit and prior to all following treatment cycles in 181 women (769 cycles) with an initial AMH level ≤1 ng/ml, undergoing IVF-ICSI. Main outcome measures were laboratory outcomes and pregnancy rates. Results Seventy patients undergoing 249 cycles had extremely low AMH levels (≤0.2 ng/ml), whereas 111 patients undergoing 520 cycles had low AMH levels (0.21–1.0 ng/ml). Number of oocytes retrieved per cycle, fertilized oocytes and number of transferred embryos were significantly lower in the extremely low AMH levels group compared to the low AMH levels (P<0.003). Crude ongoing pregnancy rates were 4.4% for both groups of patients. Among 48 cycles of women aged ≥42 with AMH levels of ≤0.2 ng/ml no pregnancies were observed. But, in patients with AMH levels of 0.2–1.0 ng/ml, 3 ongoing pregnancies out of 192 cycles (1.6%) were observed. However, in a multivariate regression analysis adjusted for age and cycle characteristics, no significant differences in ongoing pregnancy rates per cycle between the two groups were evident. Cumulative pregnancy rates of 20% were observed following five cycles, for both groups of patients. Conclusions Patients with extremely low AMH measurements have reasonable and similar pregnancy rates as patients with low AMH. Therefore, AMH should not be used as the criterion to exclude couples from performing additional IVF treatments.

Head progression distance in prolonged second stage of labor: relationship with mode of delivery and fetal head station

To evaluate the clinical significance of fetal head progression distance (HPD), measured by transperineal ultrasound, during prolonged second stage of labor.

GnRH agonist vs. hCG for triggering of ovulation--differential effects on gene expression in human granulosa cells.

Objective To investigate the mRNA expression of genes related to steroidogenesis and OHSS in granulosa cells (GCs) of patients triggered with GnRH agonist compared to patients triggered with hCG. Design Mural GCs were obtained at the time of oocyte retrieval and gene expression was analyzed using quantitative real time RT-PCR. Settings Single center, case control study. Patient(s) 24 women who were treated with GnRH agonist or hCG for triggering of ovulation. Interventions GC collection. Main Outcome Measure(s) The expression of genes related to steroidogenesis and OHSS in mural GCs Results The fertilization rate was similar in the two groups. The mRNA expression of CYP19A1 (0.50 vs 1, arbitrary unit), CYP11A1 (0.6 vs. 1) and 3 beta hydroxysteroid-dehydrogenase (0.39 vs 1) was significantly lower in the GnRH group. The expression of VEGF (0.74 vs. 1) and inhibin β B (0.38 vs 1) was lower in the GnRH analog triggered group. Conclusion Expression of genes related to steroidogenesis is lower at the time of oocyte retrieval in patients triggered with GnRH agonist. The decreased expression of VEGF and inhibin β B in the GnRH agonist group can explain the mechanism of early OHSS prevention.

Pubic arch angle in prolonged second stage of labor: clinical significance

To evaluate the clinical significance of the pubic arch angle (PAA) measured by transperineal ultrasound during prolonged second stage of labor.