A. Pellicer

Administration of Moderate and High Doses of Gonadotropins to Female Rats Increases Ovarian Vascular Endothelial Growth Factor (VEGF) and VEGF Receptor-2 Expression that Is Associated to Vascular Hyperpermeability

Abstract Convincing evidence supports the role of ovarian-origin vascular endothelial growth factor (VEGF) in inducing vascular permeability (VP) and ascites associated with ovarian hyperstimulation syndrome (OHSS) in mammals, including humans. A circulatory dysfunction has been described in every woman treated with gonadotropins for in vitro fertilization. It is not known, however, whether the action of gonadotropins also includes up-regulation of the VEGF receptor-2 (VEGFR-2) and whether increased VP is also found when milder stimulation is used. Thus, we applied an OHSS animal model to answer these questions. Immature female rats were stimulated with saline (control group) or with high (10 IU of eCG × 4 days + 30 IU hCG, OHSS group) or mild (10 IU of eCG + 10 IU of hCG, mild-stimulation group) doses of gonadotropins. The VP and the expression of whole-VEGF and VEGFR-2 mRNAs were analyzed through time-course experiments (0, 24, 48, and 96 h after hCG). Although eCG increased VP and the expression of VEGF and VEGFR-2 mRNAs in the ovaries of both mild- and OHSS-stimulated animals, hCG further augmented these parameters and produced the highest values after 48 h. A linear correlation was found between increased expression of VEGF and VEGFR-2 mRNAs and enhanced VP in both mild and OHSS groups. Immunohistochemistry showed the presence of VEGF and VEGFR-2 in the granulosa-lutein and endothelial cells of the entire corpus luteum. These studies confirm that in hyperstimulated animals as well as in mildly treated rats, VEGF and VEGFR-2 are overexpressed and associated with an increase in VP, which may be responsible for the accumulation of ascitic fluid in the syndrome.

ART and uterine pathology: how relevant is the maternal side for implantation?

BACKGROUND Assisted reproduction technology (ART) has become a standard treatment for infertile couples. Increased success rates obtained over the years have resulted primarily from improved embryo quality, but implantation rates still remain lower than expected. The uterus, an important player in implantation, has been frequently neglected. While a number of uterine pathologies have been associated with decreased natural fertility, less information exists regarding the impact of these pathologies in ART. This report reviews the evidence to help clinicians advise ART patients. METHODS An electronic search of PubMed and EMBASE was performed to identify articles in the English, French or Spanish language published until May 2014 which addressed uterine pathology and ART. Data from natural conception were used only in the absence of data from ART. Studies were classified in decreasing categories: RCTs, prospective controlled trials, prospective non-controlled trials, retrospective studies and experimental studies. Studies included in lower categories were only used if insufficient evidence was available. Pooled data were obtained from systematic reviews with meta-analyses when available. The summary of the evidence for the different outcomes and the degree of the recommendation for interventions were based on the GRADE (Grading of Recommendations Assessment, Development and Evaluation) statement recommendations. RESULTS There is strong evidence that surrogacy is effective for uterine agenesia. For the remaining pathologies, however, there is very little evidence that the established treatments improve outcomes, or that these pathologies have a negative effect on ART. In the presence of an apparently normal uterus, assessing endometrial receptivity (ER) is the goal; however diagnostic tests are still under development. CONCLUSIONS The real effect of different uterine/endometrial integrity pathologies on ART is not known. Moreover, currently proposed treatments are not based on solid evidence, and little can be done to assess ER in normal or abnormal conditions. No strong recommendations can be given based on the published experience, bringing an urgent need for well-designed studies. In this context, we propose algorithms to study the uterus in ART.

Difference in birth weight of consecutive sibling singletons is not found in oocyte donation when comparing fresh versus frozen embryo replacements.

OBJECTIVE First, to assess if there are any differences in birth weight or gestational length in newborns from egg-donation pregnancies delivering singletons, originating from either fresh or frozen-thawed embryos when they were developed and delivered within the same mothers. Second, to determine if there are any clinical, phenotypic, or laboratory factors influencing this relationship, including the origin of the oocyte (same or different donor), the order of the children (first fresh or first frozen-thawed embryo transfer), the embryo freezing technique (vitrification or slow freezing), the in vitro embryo culture length, and the duration that embryos remained frozen. DESIGN Retrospective cohorts study. SETTING University-affiliated infertility centers. PATIENT(S) A total of 360 women undergoing oocyte donation (OD), delivering (>28 weeks) at least two babies, each one from a single pregnancy, originating from at least one fresh and one frozen-thawed embryo transfer, controlling maternal and laboratory characteristics, to test the effect of embryo freezing on children size (n = 731). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Birth weight, gestational age, weight percentile, being large for gestational age (LGA), small for gestational age (SGA), size out of normal range (ONR = LGA + SGA), and macrosomy. RESULT(S) From fresh versus thawed embryos, respectively, mean birth weight of children was 3,183.7 g versus 3,226.4 g, gestational age was 272.1 days versus 268.8 days, and mean weight percentiles were 47.6 versus 50.1. The proportions and corresponding odds ratios (ORs) from fresh versus thawed embryos, respectively, were for LGA 13.6% versus 11.3% (OR 0.81), for SGA 9.4% versus 12.5% (OR 1.37), for ONR 23.1% versus 23.8% (OR 1.04), and for macrosomy 0.3% versus 0.8% (OR 3.1). After adjusting for clinically relevant variables, the ORs were for LGA 0.96, for SGA 1.40, for ONR 1.20, and for macrosomy not computable. None of the stated measures were significantly different. Also, independent analyses run on the origin of the oocytes, cryopreservation technique, cleavage stage of the embryos, and time that embryos remained frozen did not reveal any significant trends. CONCLUSION(S) This study comparing siblings from OD cycles, and eliminating the independent variables that affect early events in pregnancy, revealed no difference in duration of gestation and live birth weights between fetuses obtained after the replacement of fresh or frozen embryos. Moreover, no clinical, phenotypic, or laboratory factors appeared to be relevant, once statistically controlled.

Dopamine agonist inhibits vascular endothelial growth factor protein production and secretion in granulosa cells

BackgroundDopamine receptor 2 agonists (D2-ags) inhibit vascular endothelial growth factor (VEGF) secretion in luteinized granulosa cells (LGCs) both in vitro and in vivo. However, the mechanism of D2 regulation of the VEGF/VEGF Receptor 2 (VEGFR-2) pathway remains to be elucidated. We sought to determine the effects of D2 signaling on VEGF transcription and translation in LGCs, with the expectation of identifying potential D2-ag-based therapies for ovarian hyperstimulation syndrome (OHSS).FindingsLGCs from egg donors were cultured with chorionic gonadotropin (hCG) in the presence of Actinomycin-D (ActD) or Brefeldin-A (BFA) to evaluate the effects of a D2-ag, cabergoline (Cb2), on VEGF secretion. The contribution of the conventional Gi/Go, Gz and AKT/β-Arrestin pathways in the VEGF regulation was assessed by adding pertussis toxin (PTX), phorbol 12-myristate 13-acetate (PMA), or wortmannin (WT). While Cb2 inhibited VEGF secretion by interfering with VEGF peptide translation and secretion, inhibition of conventional D2 transduction pathways did not reverse Cb2-mediated inhibition of VEGF secretion.ConclusionsThe effects of D2-ag on VEGF translation and secretion are mediated by D2 signaling pathways that have yet to be described. We found that D2-ag inhibits VEGF secretion at the post-transcriptional level, suggesting that D2-ag treatment should be combined with therapies that inhibit VEGF transcription, such as the employment of LH or GnRH for triggering ovulation, to improve the efficacy of OHSS prevention.

Impact of ovarian stimulation with gonadotrophins on embryo aneuploidy

Sir, We have read the article published in your journal by Taylor et al. (2014) with much interest. In this review the authors properly summarize the mechanisms, origins and incidence of chromosomal mosaicism in humans. The review is rigorous, exhaustive and very well-documented, and therefore we congratulate the authors for their work. Nevertheless, we find that some relevant information about the external influences that could contribute to mosaicism and/or embryo aneuploidy is missing. Authors state that ‘hyperstimulation has been implicated in increased rates of cleavage stage aneuploidy’. We consider that this statement should not be so categorical as the potential deleterious effect of ovarian stimulation on oocyte and embryo quality is still the subject of lively debate. We conducted the first prospective cohort study by comparing unstimulated and stimulated cycles in the same woman (Labarta et al., 2012). The intrasubject comparison showed a rate of aneuploidy of 34.8% (95% CI 1⁄4 20.5–49.1) in the unstimulated cycle and 38.2% (95% CI 1⁄4 30.5–45.8) in the stimulated cycle (risk difference 1⁄4 3.4 (95% CI 1⁄4 217.9 to 11.2)). No differences were observed for embryo quality and type of chromosomal abnormalities. We concluded that ovarian stimulation does not significantly raise the embryo aneuploidy rate in IVF-derived human embryos when compared with an unstimulated cycle. The study was conducted in egg donors, younger than 35 years old with no previous ovarian stimulation treatments. The time frame between both unstimulated and stimulated cycles was a maximum of 3 months. The design of this study avoided some biases, such as impact of age on aneuploidy, intersubject variability, infertility background and different lab conditions between distinct IVF centres, such that we could analyse the net impact of gonadotrophins on embryo aneuploidy. We were also able to analyse the largest sample of IVF-derived embryos from unstimulated cycles, and we observed an aneuploidy rate of 34.8%, which is in agreement with previously published data (Verpoest et al., 2008). These results suggest that embryo aneuploidies are present in human beings, even in the absence of ovarian stimulation, and that this could be the reason why fertility in humans is so ineffective. These findings are supported by previous data showing that when comparing natural and stimulated cycles, no differences were observed in terms of cleavage capacity, embryo quality or incidence of aneuploidy in aborted fetuses. Other studies have compared the effect of different doses of gonadotrophins—instead of natural versus stimulated cycles—on the rate of mosaicism and embryo aneuploidy. In infertile patients, it has been suggested that the use of gonadotrophins might raise the chromosomal abnormality rate in a dose-dependent manner, mainly due to an increased incidence of mosaicism (Baart et al., 2007). Yet in oocyte donors, our group observed that the incidence of mosaicism wassimilar, regardless of the doses of gonadotrophins used. However, the same study showed that when doses are small, but the number of oocytes obtained is similar to higher doses in the same egg donor, the incidence of mosaicism lowers (Rubio et al., 2010). In summary, according to recent literature, it cannot be stated that ovarian stimulation with gonadotrophins may significantly increase the rate of embryo aneuploidy in patients without the negative effect of age acting as a confounding factor. Moreover, embryo aneuploidy is present even in unstimulated cycles, suggesting that either this is the real incidence in human beings or there are factors other than ovarian stimulation related with the IVF procedure which may increase this incidence in comparison with in vivo fertilization. Finally, mosaicism could be related with doses of gonadotrophins and this effect should be further studied.

Evaluation of PAI-1 in endometriosis using a homologous immunocompetent mouse model†

Abstract To analyze the role of PAI-1 (plasminogen activator inhibitor 1) in endometriotic lesion growth, we studied the effect of PAI-1 inhibition by PAI-039 using a homologous mousemodel of endometriosis that allows noninvasive monitoring. Endometrial tissue from donor mice was collected, labeled with mCherry adenovirus, and implanted into a subcutaneous pocket on the ventral abdomen of recipient mice. Seven days after transplantation, mice were randomly allocated in two groups and treated once daily for 2 weeks with either vehicle (control group) or PAI-1 inhibitor (PAI-039 group). Endometriotic lesion size generated in recipient mice was monitored by mCherry signal. Animals were euthanized 21 days after endometrial tissue implantation and endometriotic lesions were harvested for fibrin deposit and vascularization analyses. Collagen content was also examined to determine the overall effects of proteolysis on extracellular matrix degradation. We demonstrated that endometriotic lesions generated in recipient mice from both groups presented characteristics typical of human endometriotic lesions. We observed a significant decrease in fluorescence signal in endometriotic lesions from the PAI-039 group at the beginning of the treatment correlated with a decrease in endometriotic lesion size. PAI-1 inhibition significantly decreased lesion cell proliferation. In addition, endometriotic lesions from the PAI-1 inhibition group showed a decreased percentage of neovascularization as well as fibrin deposits. However, the density and distribution of collagen were not affected by PAI-039. Our results suggest that in vivo inhibition of PAI-1 by PAI-039 may be a useful strategy to reduce endometriotic lesion size by blocking angiogenesis. Summary Sentence PAI-1 inhibition reduces angiogenesis and decreases endometriotic lesion size in a homologous mouse model of endometriosis.

The timing of administration of letrozole significantly affects the oocyte recovery rate in breast cancer patients undergoing controlled ovarian stimulation for fertility preservation

THE TIMING OF ADMINISTRATION OF LETROZOLE SIGNIFICANTLYAFFECTS THE OOCYTE RECOVERY RATE IN BREAST CANCER PATIENTS UNDERGOING CONTROLLED OVARIAN STIMULATION FOR FERTILITY PRESERVATION. C. Diaz-Garcia, J. Domingo, A. Romero, M. Martinez, J. M. Rubio, J. A. Garcia-Velasco, A. Pellicer. Woman’s Health Area, La Fe University Hospital, Valencia, Spain; IVI-Las Palmas, Las Palmas de Gran Canaria, Spain; IVIMadrid,Madrid, Spain; La Fe University Hospital, Valencia, Spain; IVI-Valencia, Valencia, Spain.