Daniela Galliano

MicroRNA and implantation

We provide a review of microRNA (miRNA) related to human implantation which shows the potential diagnostic role of miRNAs in impaired endometrial receptivity, altered embryo development, implantation failure after assisted reproduction technology, and in ectopic pregnancy and pregnancies of unknown location. MicroRNAs may be emerging diagnostic markers and potential therapeutic tools for understanding implantation disorders. However, further research is needed before miRNAs can be used in clinical practice for identifying and treating implantation failure.

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.

A new more detailed stratification of low responders to ovarian stimulation: from a poor ovarian response to a low prognosis concept

The management of patients with impaired or poor ovarian response (POR) remains a controversial and complex clinical issue. A systematic review of 47 randomized controlled trials revealed 41 different definitions of POR (1). Notably, the number of oocytes retrieved was adopted as a criterion of POR in 40% of the trials, although the threshold number differed considerably among studies (1). To standardize the definition of POR, Ferraretti et al. (2) proposed new criteria, known as the ‘‘Bologna criteria,’’ based on three conditions: 1) advanced maternal age (R40 years) or any other POR risk factor; 2) a previous incident of POR; and 3) a low ovarian reserve test in terms of antim€ ullerian hormone (AMH) and antral follicle count (AFC). Two of these three criteria are required for a POR diagnosis. In addition, two cycles with POR after maximal stimulation are sufficient to classify a patient as a poor responder even in the absence of the other criteria mentioned. Although the Bologna criteria were found to be useful in predicting the outcome of IVF and for counseling purposes, their use in clinical trials has been questioned because they entail the risk of grouping together women who differ significantly in biologic characteristics (3). For example, according to the Bologna criteria, young women with a low ovarian reserve associated with a previous episode of POR, young women with a normal ovarian reserve and two POR episodes, and older women (R40 years) with a normal ovarian reserve and a previous episode of POR would be included in the same category even though the clinical management of these patients requires different strategies. In clinical terms, apart from the number of oocytes retrieved, various features that may affect treatment outcomes must be considered in the management of patients, namely: 1) the age-related embryo/blastocyst aneuploidy rate, which could dramatically change the prognosis in women that have the same oocyte yield; and 2) ovarian ‘‘sensitivity’’ to exogenous gonadotropins, which could be related to a specific genetic profile. To introduce a more nuanced picture of POR, we here propose clinically relevant criteria that can help to guide the physician in the management of patients. In detail, we suggest a more specific new definition of ‘‘low prognosis’’ patients that:

Female obesity: short- and long-term consequences on the offspring

Abstract The worldwide prevalence of obesity has risen over the past few decades and women are currently more likely than ever to enter pregnancy obese. Pre-pregnancy obesity and excessive gestational weight gain increase miscarriage rates and obstetric and neonatal complications, which result in a lower healthy live birth rate. In addition to its negative consequences for the mother, obesity has been shown to be an important risk factor for chronic illnesses, such as cardiovascular disease, metabolic syndrome and type 2 diabetes in the adolescence and adulthood of the offspring. Moreover, maternal obesity causes psychological problems, physical disabilities and higher healthcare costs. Fetal programming of metabolic function induced by obesity, through physiological and/or epigenetic mechanisms, may have an intergenerational effect and could, thus, perpetuate obesity in the next generation. In order to break this vicious circle and avoid serious short- and long-term negative outcomes for both mothers and fetuses, the prevention and adequate management of obesity and gestational weight gain are essential.

Blastocyst development in single medium with or without renewal on day 3: a prospective cohort study on sibling donor oocytes in a time-lapse incubator

OBJECTIVE To evaluate the efficiency of using a continuous (one-step) protocol with a single medium for the culture of human embryos in a time-lapse incubator (TLI). DESIGN Prospective cohort study on sibling donor oocytes. SETTING University-affiliated in vitro fertilization (IVF) center. PATIENT(S) Embryos from 59 patients. INTERVENTION(S) Culture in a TLI in a single medium with or without renewal of the medium on day-3. MAIN OUTCOME MEASURE(S) Embryo morphology and morphokinetic parameters, clinical pregnancy, take-home baby rate, and perinatal outcomes. RESULT(S) The blastocyst rates (68.3 vs. 66.8%) and the proportion of good-quality blastocysts (transferred plus frozen) obtained with the two-step (80.0%) protocol were statistically significantly similar to those obtained in the one-step protocol (72.2%). Similarly, morphokinetic events from early cleavage until late blastocyst stages were statistically significantly equivalent between both groups. No differences were found either in clinical pregnancy rates when comparing pure transfers performed with embryos selected from the two-step (75.0%), one-step (70.0%, respectively), and mixed (57.1%) groups. A total of 55 out of 91 embryos transferred implanted successfully (60.4%), resulting in a total of 37 newborns with a comparable birth weight mean among groups. CONCLUSION(S) Our findings support the idea that in a TLI with a controlled air purification system, human embryos can be successfully cultured continuously from day 0 onward in single medium with no need to renew it on day-3. This strategy does not affect embryo morphokinetics or development to term and offers more stable culture conditions for embryos as well as practical advantages and reduced costs for the IVF laboratory.

Day-3 embryo metabolomics in the spent culture media is altered in obese women undergoing in vitro fertilization

OBJECTIVE To determine whether the global metabolomic profile of the spent culture media (SCM) of day-3 embryos is different in obese and normoweight women undergoing in vitro fertilization (IVF). DESIGN Prospective cohort analysis. SETTING IVF clinic. PATIENT(S) Twenty-eight young, nonsmoking women with normoweight, nonsmoking male partners with mild/normal sperm factors undergoing a first IVF attempt for idiopathic infertility, tubal factor infertility, or failed ovulation induction: obese ovulatory women (n = 12); obese women with polycystic ovary syndrome (PCOS; n = 4); normoweight ovulatory women (n = 12). INTERVENTION(S) Fifty μl of SCM collected from two day-3 embryos of each cohort. MAIN OUTCOME MEASURE(S) Metabolomic profiling via ultrahigh performance liquid chromatography coupled to mass spectrometry of SCM from a total of 56 embryos. RESULT(S) The untargeted metabolomic profile was different in obese and normoweight women. Partial least squares discriminant analysis resulted in a clear separation of samples when a total of 551 differential metabolites were considered. A prediction model was generated using the most consistent metabolites. Most of the metabolites identified were saturated fatty acids, which were detected in lower concentrations in the SCM of embryos from obese women. The metabolomic profile was similar in obese women with or without PCOS. CONCLUSION(S) The metabolomic profile in the SCM of day-3 embryos is different in normoweight and obese women. Saturated fatty acids seem to be reduced when embryos from obese patients are present. CLINICAL TRIAL REGISTRATION NUMBER NCT01448863.

Successful pregnancy after treatment with ulipristal acetate for uterine fibroids.

This case report presents a clinical pregnancy after ulipristal acetate (UA) to decrease uterine fibroid size. A 37-year-old patient, gravida 1, abortus 1, with uterine fibroids was treated with 5 mg of UA daily for 13 weeks starting eight months after a multiple laparotomic myomectomy. Fibroid shrinkage and restoration of the morphology of endometrial cavity were evaluated in order to allow a subsequent pregnancy. A decrease of the uterine fibroids and a normal morphology of the endometrial cavity were noted by transvaginal ultrasound after treatment. An endometrial biopsy excluded histologic endometrial changes. Three months after the end of UA the patient reported amenorrhea for 5 weeks and a clinical pregnancy was confirmed with transvaginal ultrasound. She underwent a subsequent uneventful pregnancy. Thus, the spontaneous pregnancy after UA to reduce fibroid size may support the potential clinical utility of this selective progesterone receptor modulator in the management of women with pregnancy desire and uterine fibroids after a prior myomectomy. Patients who refuse a new surgical procedure and/or those who are going to undergo assisted reproductive techniques would benefit from UA. It effectively shrinks fibroids, avoids risks of a new surgical procedure, and allows an immediate attempt at conception after the end of treatment.

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.

Optimal uterine anatomy and physiology necessary for normal implantation and placentation

The authors review aberrations of uterine anatomy and physiology affecting pregnancy outcomes with IVF. In the case of endometriosis and hydrosalpinx, pathologies outside of the uterus alter the uterine endometrium. In the case of endometriosis, Dominique de Ziegler outlines the numerous changes in gene expression and the central role of inflammation in causing progesterone resistance. With endometriosis, the absence of ovarian function inherent in deferred transfer, with or without a more lengthy suppression of ovarian function, appears to be sufficient to restore normal function of eutopic endometrium. Because laparoscopy is no longer routine in the evaluation of infertility, unrecognized endometriosis then becomes irrelevant in the context of assisted reproductive technology. With hydrosalpinx and submucus myomas, the implantation factor HOXA-10 is suppressed in the endometrium and, with myomas, even in areas of the uterus not directly affected. Daniela Galliano reviews various uterine pathologies, the most enigmatic being adenomyosis, where the endometrium also manifests many of the changes seen in endometriosis and deferred transfer with extended suppression appears to provide the best outcomes. Ettore Cicinellis group has extensively studied the diagnosis and treatment of endometritis, and although more definitive diagnosis and care of this covert disorder may await techniques such as sequencing of the endometrial microbiome, it undoubtedly is an important factor in implantation failure, deserving our attention and treatment.

Introduction: MicroRNAs in human reproduction: small molecules with crucial regulatory roles

MicroRNAs constitute a large family of approximately 21-nucleotide-long, noncoding RNAs. They emerged more than 20 years ago as key posttranscriptional regulators of gene expression. The regulatory role of these small RNA molecules has recently begun to be explored in the human reproductive system. In this issues Views and Reviews, the authors present the current knowledge regarding the involvement of microRNAs in several aspects of human reproduction and discuss its future implications for clinical practice.