Ramon Carreras

Fresh embryo transfer versus frozen embryo transfer in in vitro fertilization cycles: a systematic review and meta-analysis

OBJECTIVE To examine the available evidence to assess if cryopreservation of all embryos and subsequent frozen embryo transfer (FET) results in better outcomes compared with fresh transfer. DESIGN Systematic review and meta-analysis. SETTING Centers for reproductive care. PATIENT(S) Infertility patient(s). INTERVENTION(S) An exhaustive electronic literature search in MEDLINE, EMBASE, and the Cochrane Library was performed through December 2011. We included randomized clinical trials comparing outcomes of IVF cycles between fresh and frozen embryo transfers. MAIN OUTCOME MEASURE(S) The outcomes of interest were ongoing pregnancy rate, clinical pregnancy rate, and miscarriage. RESULT(S) We included three trials accounting for 633 cycles in women aged 27-33 years. Data analysis showed that FET resulted in significantly higher ongoing pregnancy rates and clinical pregnancy rates. CONCLUSION(S) Our results suggest that there is evidence that IVF outcomes may be improved by performing FET compared with fresh embryo transfer. This could be explained by a better embryo-endometrium synchrony achieved with endometrium preparation cycles.

Two new single-nucleotide polymorphisms in the COL1A1 upstream regulatory region and their relationship to bone mineral density.

Single‐nucleotide polymorphisms (SNPs) in regulatory regions of candidate genes may determine variability in bone mineral density (BMD) because they may be responsible for differences in levels of a gene product in response to external signals. Under this hypothesis, we scanned an 800‐base pair (bp) region within the COL1A1 promoter, known to harbor cis elements important for in vivo expression, and we found two new polymorphisms: −1663indelT and −1997 G/T. The G to T transversion at −1997 was associated with lumbar spine BMD (p = 0.015) when tested in a cohort of 256 postmenopausal women after adjusting by age, body weight, and years since menopause; a lower degree of association was detected also for femoral neck BMD in a subgroup of 146 women in univariate analysis and after adjusting by age (p = 0.044). The polymorphism −1663indelT, which corresponds to a deletion of a T in a tract of eight T residues (−1670 to −1663), did not show significant association with BMD. Interestingly, −1663indelT is in strong linkage disequilibrium (LD) with the previously described Sp1 polymorphism of intron 1, which in this study did not show association with BMD either. Significant interaction between −1997 G/T and −1663indelT (p = 0.019), and between −1997 G/T and Sp1 (p = 0.045) was observed also. Individuals heterozygous for the three polymorphisms showed the highest mean BMD value. Gel retardation assays showed that oligonucleotides containing either the −1663 or the −1997 polymorphic sites specifically bind primary osteoblast nuclear proteins. We named these binding sites as PCOL1 and PCOL2, respectively. In summary, this study describes two new SNPs in the COL1A1 promoter, which may affect bone mass determination.

Simvastatin and atorvastatin enhance gene expression of collagen type 1 and osteocalcin in primary human osteoblasts and MG‐63 cultures

To clarify the mechanism of the stimulatory effect of statins on bone formation, we have assessed the effect of simvastatin and atorvastatin on osteoblast activity by analysing cell proliferation, as well as collagen, osteocalcin, and bone morphogenetic protein‐2 (BMP2) gene expression in primary human osteoblast (hOB) and MG‐63 cell line cultures. Explants of bone from patients without any metabolic disease under orthopedic hip procedures were used to obtain hOB. Cell cultures were established, synchronized, and different concentrations of simvastatin or atorvastatin were added (10−9 M, 10−8 M, 10−7 M, 10−6 M) during the experiment. Cell proliferation was analyzed after 24 h. Collagen polypeptide α1 type 1 (COL1A1) gene expression, osteocalcin, and BMP2 expression levels were quantified by real‐time PCR after 24 h incubation with statins. There was a statistically significant decrease in cell proliferation related to simvastatin or atorvastatin addition at all concentrations in primary hOB compared with those not treated. A significant increase in COL1A1, osteocalcin, and BMP2 gene expression was detected when hOB cultures were treated with simvastatin or atorvastatin at different concentrations. Similar but less significant effects were found on MG‐63 cells. After statin treatment we observed both an arrest of proliferation in hOB cells and an increase in collagen, osteocalcin, and BMP2 gene expression, consistent with a stimulatory effect towards mature osteoblast differentiation. These findings support the bone‐forming effect of statins, probably through the BMP2 pathway. J. Cell. Biochem. 101: 1430–1438, 2007.

Molecular markers of endometrial carcinoma detected in uterine aspirates.

Endometrial cancer (EC) is the most frequent of the invasive tumors of the female genital tract. Although usually detected in its initial stages, a 20% of the patients present with advanced disease. To date, no characterized molecular marker has been validated for the diagnosis of EC. In addition, new methods for prognosis and classification of EC are needed to combat this deadly disease. We thus aimed to identify new molecular markers of EC and to evaluate their validity on endometrial aspirates. Gene expression screening on 52 carcinoma samples and series of real‐time quantitative PCR validation on 19 paired carcinomas and normal tissue samples and on 50 carcinoma and noncarcinoma uterine aspirates were performed to identify and validate potential biomarkers of EC. Candidate markers were further confirmed at the protein level by immunohistochemistry and Western blot. We identified ACAA1, AP1M2, CGN, DDR1, EPS8L2, FASTKD1, GMIP, IKBKE, P2RX4, P4HB, PHKG2, PPFIBP2, PPP1R16A, RASSF7, RNF183, SIRT6, TJP3, EFEMP2, SOCS2 and DCN as differentially expressed in ECs. Furthermore, the differential expression of these biomarkers in primary endometrial tumors is correlated to their expression level in corresponding uterine fluid samples. Finally, these biomarkers significantly identified EC with area under the receiver‐operating‐characteristic values ranging from 0.74 to 0.95 in uterine aspirates. Interestingly, analogous values were found among initial stages. We present the discovery of molecular biomarkers of EC and describe their utility in uterine aspirates. These findings represent the basis for the development of a highly sensitive and specific minimally invasive method for screening ECs.

Polymorphisms in the interleukin-6 receptor gene are associated with bone mineral density and body mass index in Spanish postmenopausal women

OBJECTIVE Osteoporosis and obesity are complex diseases with a strong genetic component. Bone mineral density (BMD) and body mass index (BMI) linkage studies identified a locus at 1q21-23, where the interleukin-6 receptor (IL6R) gene is located. The IL6R and the gp130 receptors are the mediators of IL6 action. Serum levels of IL6 and sIL6R (the soluble form of IL6R) are higher in several diseases such as osteoporosis or obesity. Variants at IL6R have been associated with BMI and obesity. However, IL6R is an as-yet-unexplored osteoporosis candidate gene. DESIGN In the present study we analysed two polymorphisms in the IL6R promoter, -1435 C/T (rs3887104) and -208 G/A (rs4845617), and the Asp358Ala polymorphism (rs8192284), in relation to both BMD and BMI in a cohort of 559 postmenopausal Spanish women. RESULTS The promoter polymorphisms, -1435 C/T and -208 G/A were associated with femoral neck (FN) BMD (P=0.011 and P=0.025 respectively). The C-A and T-G promoter haplotypes were also associated with FN BMD. Additionally, the Asp358Ala variant was associated with lumbar spine BMD (P=0.038). Finally, the -208 G/A polymorphism and the C-G and C-A haplotypes were associated with BMI and obesity, where GG was the risk genotype (P=0.033 for BMI; P=0.010 for obesity). CONCLUSION These data suggest that variants in the IL6R gene are not only involved in the determination of BMI but also relevant for the determination of BMD. The IL6R gene may belong to the growing list of genes known to be involved in both phenotypes.

Proteomic approach to ETV5 during endometrial carcinoma invasion reveals a link to oxidative stress

Endometrial cancer, the most common gynecological malignancy in western countries, is characterized by a favorable prognosis. Nonetheless, deep myometrial invasion correlates with more undifferentiated tumors, lymph-vascular invasion, node involvement and decreased global survival. We have described previously the Ets family member ERM/ETV5 specifically upregulated in endometrial endometrioid carcinoma (EEC) associated with myometrial infiltration. To understand the role of this transcription factor during myometrial infiltration, we analyzed by two-dimension differential gel electrophoresis (2D-DIGE) technology those proteins whose expression was altered in endometrial cell lines stably overexpressing ERM/ETV5. Pathway analysis pointed to actin regulation and transforming growth factor beta and progesterone signaling as processes regulated by ERM/ETV5. In addition, we characterized the specific upregulation of the nuclear dehydrogenase/reductase Hep27 as well as its ERM/ETV5-dependent mitochondrial localization. Further functional studies demonstrated a protective role of Hep 27 against apoptosis induced by oxidative stress. Overall, the ETV5-related proteomic approach performed in the Hec-1A cell line reinforces a role of this transcription factor in the regulation of the migratory and invasive tumor behavior and points to a modulated response to oxidative stress associated with the promotion of invasion in endometrial cancer. Unraveling the molecular events in EEC associated with the initiation of tumor invasion would represent an obvious improvement in the pursuit of rational targets for the onset of metastasis. This knowledge would also be a valuable tool for the molecular stratification of patients since myometrial affectation determines an increase in the rate of recurrence after a first surgical treatment and a decrease in 5 year survival.

IVF/ICSI with or without preimplantation genetic screening for aneuploidy in couples without genetic disorders: a systematic review and meta-analysis

PurposeTo assess the efficacy of preimplantation genetic screening to increase ongoing pregnancy rates in couples without known genetic disorders.MethodsSystematic review and meta-analysis of randomized controlled trials. Two reviewers independently determined study eligibility and extracted data.ResultsTen randomized trials (1,512 women) were included. The quality of evidence was moderate. Meta-analyses using a random-effects model suggest that PGS has a lower rate of ongoing pregnancies (risk ratio=0.73, 95% confidence interval 0.62–0.87) and a lower rate of live births (risk ratio=0.76, 95% confidence interval 0.64–0.91) than standard in vitro fertilization/intracytoplasmic sperm injection.ConclusionsIn women with poor prognosis or in general in vitro fertilization program, in vitro fertilization/intracytoplasmic sperm injection with preimplantation genetic screening for aneuploidy does not increase but instead was associated with lower rates of ongoing pregnancies and live births. The use of preimplantation genetic screening in daily practice does not appear to be justified.

Promoter 2 -1025 T/C Polymorphism in the RUNX2 Gene Is Associated with Femoral Neck BMD in Spanish Postmenopausal Women

Stimulation of bone formation is a key therapeutic target in osteoporosis. Runx2 is a runt domain transcription factor essential to osteoblast differentiation, bone remodeling, and fracture healing. Runx2 knockout mice exhibit a complete lack of ossification, while overexpression of this gene in transgenic mice results in an osteoporotic phenotype. Thus, RUNX2 is a good candidate for the genetic determination of osteoporosis. In this association study, the effects of the -330 G/T polymorphism in promoter 1 and the -1025 T/C polymorphism (rs7771980) in promoter 2 of RUNX2 were tested in relation to lumbar spine (LS) and femoral neck (FN) bone mineral density (BMD) in a cohort of 821 Spanish postmenopausal women. The minor allele frequencies for the two polymorphisms were 0.15 and 0.07, respectively. The two polymorphisms, located more than 90 kb apart, were not in linkage disequilibrium (D′ = 0.27, r2 = 0.028). In an ANCOVA test adjusting by weight, height, age, and years since menopause, the -330 G/T polymorphism was not associated with any of the phenotypes analyzed, while we found the -1025 T/C polymorphism to be associated with FN BMD (p = 0.001). In particular, individuals carrying the TC genotype had higher mean adjusted FN BMD values than those bearing the TT genotype. Our results highlight the importance of this RUNX2 promoter 2 polymorphism in FN BMD determination.

3q26 (hTERC) gain studied by fluorescence in situ hybridization as a persistence-progression indicator in low-grade squamous intraepithelial lesion cases

Gains of 3q26 chromosome region, where the human telomerase RNA gene (hTERC) is located, have been previously documented in cervical carcinomas and preneoplastic lesions. The aim of our study was to define the value of 3q26 gains related to persistence-progression in cervical specimens with cytologic diagnosis for low-grade squamous intraepithelial lesions, using liquid-based cytology (ThinPrep; Hologic, Marlborough, MA) and fluorescence in situ hybridization. For these purposes, 55 patients were included in the study: 25 cases with a negative cytologic diagnosis for squamous intraepithelial lesion or malignancy (20 premenopausal and 5 postmenopausal women, used as control negative cases) and 30 low-grade squamous intraepithelial lesion cases. The follow-up was performed using cytology at 6, 12, and 24 months after the low-grade squamous intraepithelial lesion diagnosis. When the cytology result showed a high-grade lesion, colposcopy and biopsy were performed. Fluorescence in situ hybridization technique with a 3q26 2-color commercial probe was performed to determine the number of hTERC copies. There were no differences between premenopausal and postmenopausal normal cases. Low-grade squamous intraepithelial lesion cases with regression in the follow-up at 6, 12, and 24 months showed a percentage of cells with 3q26 gains similar to the control cases and lower than low-grade squamous intraepithelial lesion cases with persistence or progression (P < .05). Fluorescence in situ hybridization results were similar in preserved and frozen samples. However, in frozen samples, the number of cells suitable to be evaluated by fluorescence in situ hybridization was lower than in preserved (nonfrozen) cases. In conclusion, the determination by fluorescence in situ hybridization of 3q26 gains in low-grade squamous intraepithelial lesion cases could be useful to predict the persistence-progression of such cervical lesions using both preserved and frozen cervical material.

Effects of transdermal testosterone in poor responders undergoing IVF: systematic review and meta-analysis

A systematic review and meta-analysis was performed to evaluate the effect of transdermal testosterone preceding ovarian stimulation in women with poor ovarian response undergoing IVF. Studies comparing pretreatment with transdermal testosterone versus standard ovarian stimulation among poor responders were included. The main outcome assessed was live birth. Three trials were included (113 women in the testosterone group, 112 in the control group). Testosterone-treated women achieved significantly higher live birth rate (risk ratio, RR, 1.91, 95% CI 1.01 to 3.63), clinical pregnancy rate (RR 2.07, 95% CI 1.13 to 3.78) and required significantly lower doses of FSH (RR -461.96, 95% CI -611.82 to -312.09). However, differences observed in clinical pregnancy per embryo transferred were not statistically significant (RR 1.72, 95% CI 0.91 to 3.26). No differences were observed regarding number and quality of the oocytes retrieved. In conclusion, transdermal testosterone significantly increases live birth and reduces the doses of FSH required. These findings support the theoretical synergistic role of androgens and FSH on folliculogenesis. The present data should be interpreted with caution because of the small number of trials and clinical heterogeneity. The identification of poor responders that could especially benefit from testosterone treatment should be addressed in further studies. The poor response to ovarian stimulation among women undergoing IVF is of great concern in reproductive medicine. Certain modalities have been tested to improve this response to gonadotrophin stimulation, although results from some studies have shown conflicting results. Hence, a systematic review and meta-analysis was performed in order to evaluate the effect of transdermal testosterone prior to ovarian stimulation among these women with poor ovarian response. The main outcome assessed was live birth rate. In all, three trials were included, which comprehended 113 women in the testosterone group and 112 in the control group. Women that were pretreated with transdermal testosterone achieved significantly higher live birth rate and clinical pregnancy rate and required significantly lower doses of exogenous FSH as compared with controls. However, when clinical pregnancy rate was adjusted per embryo transferred differences observed were not statistically significant. No differences were observed in the number and quality of the oocytes retrieved. In conclusion, transdermal testosterone prior to ovarian stimulation significantly increases live birth and reduces the doses of FSH required among poor responders. In addition, the identification of poor responders that could especially benefit from testosterone treatment should be addressed in further studies.