6Artificial shrinkage before fresh blastocyst transfer and IVF outcomes: a pilot randomized controlled study.

RESEARCH QUESTION: Does artificial shrinkage before fresh blastocyst transfer improve clinical pregnancy rates in IVF? DESIGN: In this monocentric prospective, randomized, double-blind, controlled pilot study, 150 couples undergoing fresh single-blastocyst transfer were randomized between 20 May 2018 and 22 February 2022. In the artificial shrinkage group (AS group), a single laser pulse was directed to the cellular junction of the trophectoderm on the opposite side of the inner cell mass in each blastocyst. IVF outcomes were clinical pregnancy, multiple pregnancy and live birth rates. Cell-free DNA (cfDNA) concentration was also measured by quantitative real-time PCR in the blastocyst culture medium. RESULTS: In total, 142 couples underwent fresh single-blastocyst transfer: control group, no artificial shrinkage, n = 47; and AS group, artificial shrinkage, n = 95; An intention-to-treat (ITT) analysis was employed. After a reassessment and the exclusion of patients with major protocol deviations, 139 couples underwent fresh single-blastocyst transfer under optimal conditions: control group, n = 47; and AS group, n = 92; a per-protocol analysis was used here. The clinical and laboratory characteristics were not significantly different between the groups. The clinical pregnancy rate was similar in the control and AS groups (ITT: 48.9% versus 49.5%, P = 0.97; per protocol: 48.94% versus 51.1%, P = 0.89). The multiple pregnancy rate and the live birth rate were also similar between the groups. No significant differences in gestational age, birthweight or proportion of male/female newborns were observed. The concentration of cfDNA in the blastocyst culture medium was not associated with IVF outcome. CONCLUSIONS: Large-scale randomized controlled trials are required to confirm these preliminary results.

Transgenerational Transmission of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Effects in Human Granulosa Cells: The Role of MicroRNAs.

Endocrine-disrupting chemicals (EDCs) might contribute to the increase in female-specific cancers in Western countries. 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) is considered the "prototypical toxicant" to study EDCs' effects on reproductive health. Epigenetic regulation by small noncoding RNAs (sncRNAs), such as microRNAs (miRNA), is crucial for controlling cancer development. The aim of this study was to analyze transcriptional activity and sncRNA expression changes in the KGN cell line after acute (3 h) and chronic (72 h) exposure to 10 nM TCDD in order to determine whether sncRNAs' deregulation may contribute to transmitting TCDD effects to the subsequent cell generations (day 9 and day 14 after chronic exposure). Using Affymetrix GeneChip miRNA 4.0 arrays, 109 sncRNAs were found to be differentially expressed (fold change < -2 or >2; p-value < 0.05) between cells exposed or not (control) to TCDD for 3 h and 72 h and on day 9 and day 14 after chronic exposure. Ingenuity Pathway Analysis predicted that following the acute and chronic exposure of KGN cells, sncRNAs linked to cellular development, growth and proliferation were downregulated, and those linked to cancer promotion were upregulated on day 9 and day 14. These results indicated that TCDD-induced sncRNA dysregulation may have transgenerational cancer-promoting effects.

Molecular Characterization of a Rare Case of Monozygotic Dichorionic Diamniotic Twin Pregnancy after Single Blastocyst Transfer in Preimplantation Genetic Testing (PGT).

Preimplantation genetic testing (PGT) is widely used to select unaffected embryos, increasing the odds of having a healthy baby. During the last few decades, it was accepted that monozygotic dichorionic diamniotic twin pregnancies occurred from the embryo splitting before Day 3 postfertilization according to Corner's dogma. Hence, the occurrence of a dichorionic diamniotic twin pregnancy after a single blastocyst transfer was considered a dizygotic pregnancy resulting from blastocyst transfer and concurrent natural fertilization. In our study, we have provided for the first time molecular proof that a single blastocyst transfer can result in a monozygotic dichorionic diamniotic twin pregnancy, invalidating Corner's dogma. In this case, we recommend systematically assessing the genetic status of dichorionic twins after single blastocyst transfer using prenatal diagnosis to exclude the risk from a potential concurrent spontaneous pregnancy and to ensure that both fetuses are unaffected. To achieve this goal, we have developed here an innovative noninvasive prenatal diagnosis by exclusion of paternal variants with droplet digital PCR, maximizing the reliability of genetic diagnosis. Further multicentric prospective studies using genetic testing are now required to establish the rate of blastocyst splitting leading to dichorionic pregnancy in PGT and to identify the risk factors.

Diethylstilbestrol exposure during pregnancy with primary clear cell carcinoma of the cervix in an 8-year-old granddaughter: a multigenerational effect of endocrine disruptors?

To date, vaginal/cervical clear cell adenocarcinoma (CCAC) has not been reported in the granddaughters of women treated with diethylstilbestrol (DES) during pregnancy. We present an 8-year-old girl with a history of severe vaginal bleeding who was diagnosed with cervical CCAC. She underwent fertility-sparing surgery and radiotherapy. No sign of recurrence was detected throughout a 10-year follow-up. Her grandmother had received DES therapy during pregnancy with the patient's mother. Although no direct causal link is demonstrated, this case raises for the first time, the hypothesis of multigenerational effects of DES in girls and strongly suggests the need to follow the granddaughters of DES-treated women.

Multigenerational endometriosis : consequence of fetal exposure to diethylstilbestrol ?

BACKGROUND: Endometriosis, which affects 10-15 % of women of reproductive age, is an estrogen-driven condition influenced by environmental and genetic factors. Exposition to estrogen-like endocrine-disrupting chemicals (EDCs) has been reported to contribute to the fetal origin of this disease. CASE PRESENTATION: We report here an informative family in which all prenatally DES-exposed daughters and subsequent granddaughters presented endometriosis, whereas the unexposed first daughter and her progeny presented no gynecological disorders. Moreover, the only post-pubertal great-granddaughter, who presents chronic dysmenorrhea that remains resistant to conventional therapy, is at risk of developing endometriosis. The mother (I-2) was prescribed DES (30 mg/day for 3 months) to inhibit lactation after each delivery. CONCLUSIONS: Although a direct causal link between the grandmother's treatment with DES and the development of endometriosis in possibly three exposed generations remains speculative, this report strengthens the suspicion that fetal exposition to DES contributes to the pathogenesis of adult diseases, such as endometriosis. It also highlights a multigenerational and likely transgenerational effect of EDCs.

Experimental Evidence of 2,3,7,8-Tetrachlordibenzo-p-Dioxin (TCDD) Transgenerational Effects on Reproductive Health.

Previous studies have demonstrated that endocrine disruptors (EDs) can promote the transgenerational inheritance of disease susceptibility. Among the many existing EDs, 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) affects reproductive health, including in humans, following direct occupational exposure or environmental disasters, for instance the Agent Orange sprayed during the Vietnam War. Conversely, few studies have focused on TCDD multigenerational and transgenerational effects on human reproductive health, despite the high amount of evidence in animal models of such effects on male and female reproductive health that mimic human reproductive system disorders. Importantly, these studies show that paternal ancestral TCDD exposure substantially contributes to pregnancy outcome and fetal health, although pregnancy outcome is considered tightly related to the woman's health. In this work, we conducted a systematic review of the literature and a knowledge synthesis in order (i) to describe the findings obtained in rodent models concerning TCDD transgenerational effects on reproductive health and (ii) to discuss the epigenetic molecular alterations that might be involved in this process. As ancestral toxicant exposure cannot be changed in humans, identifying the crucial reproductive functions that are negatively affected by such exposure may help clinicians to preserve male and female fertility and to avoid adverse pregnancy outcomes.

Is cell-free DNA in spent embryo culture medium an alternative to embryo biopsy for preimplantation genetic testing? A systematic review.

Preimplantation genetic testing (PGT) is increasingly used worldwide. It currently entails the use of invasive techniques, i.e. polar body, blastomere, trophectoderm biopsy or blastocentesis, to obtain embryonic DNA, with major technical limitations and ethical issues. Evidence suggests that invasive PGT can lead to genetic misdiagnosis in the case of embryo mosaicism, and, consequently, to the selection of affected embryos for implantation or to the destruction of healthy embryos. Recently, spent culture medium (SCM) has been proposed as an alternative source of embryonic DNA. An increasing number of studies have reported the detection of cell-free DNA in SCM and highlighted the diagnostic potential of non-invasive SCM-based PGT for assessing the genetic status of preimplantation human embryos obtained by IVF. The reliability of this approach for clinical applications, however, needs to be determined. In this systematic review, published evidence on non-invasive SCM-based PGT is presented, and its current benefits and limitations compared with invasive PGT. Then, ways of optimizing and standardizing procedures for non-invasive SCM-based PGT to prevent technical biases and to improve performance in future studies are discussed. Finally, clinical perspectives of non-invasive PGT are presented and its future applications in reproductive medicine highlighted.

The reproductive microbiome - clinical practice recommendations for fertility specialists.

The interest in and understanding of the human microbiome has grown remarkably over recent years. Advances in molecular techniques have allowed researchers to identify and study the microbiota and also use this information to develop therapeutic solutions for a spectrum of conditions. Alongside the growing interest in the microbiome, societal changes have resulted in many couples looking to start families later in life, therefore increasing the demand for assisted reproductive technologies. Combining these trends, it makes sense that clinicians are eager to understand and exploit the microbiome of their patients, i.e. the reproductive microbiome, in order to help them achieve their goal of becoming parents. This paper aims to provide an overview of the current and future research into the reproductive microbiome in relation to fertility and also share clinical practice recommendations for physicians who are new to this field or unsure about how they can utilise what is known to help their patients.

Prokineticin 1 is a new biomarker of human oocyte competence: expression and hormonal regulation throughout late folliculogenesis.

CONTEXT: Prokineticin 1 (PROK1) quantification in global follicular fluid (FF) has been recently reported as a predictive biomarker of in vitro fertilization (IVF) outcome. It is now necessary to evaluate its clinical usefulness in individual follicles. OBJECTIVES: To evaluate the clinical value of PROK1 secretion in individual FF to predict oocyte competence. To determine the impact of follicular size, oocyte maturity, and gonadotropin treatments on PROK1 secretion. DESIGN AND SETTING: Prospective cohort study from May 2015 to May 2017 at the University Hospital of Grenoble. PATIENTS: A total of 69 infertile couples underwent IVF. INTERVENTION(S): Collection of 298 individual FF from 44 women undergoing IVF; 52 individual cumulus cell (CC) samples and 15 CC primary cultures from 25 women undergoing IVF-intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Oocyte competence was defined as the ability to sustain embryo development to the blastocyst stage. Follicular size was measured by 2D-sonography. PROK1 concentration was quantified by ELISA assay. RESULTS: PROK1 concentration was correlated to follicular size (r = 0.85, P = 2.2 × 10-16). Normalized PROK1 concentration in FF was predictive of subsequent oocyte competence (AUROC curve = 0.76 [95% CI, 0.69-0.83]; P = 1.7 × 10-9), irrespectively of day-2 embryo morphokinetic parameters. The expression and secretion of PROK1 were increased in FF and CC of mature oocytes (P < 0.01). Follicle Stimulating Hormone and hCG up-regulated PROK1 secretion in CC primary cultures (P < 0.01; P < 0.05), probably through the cAMP pathway (P < 0.01). CONCLUSIONS: PROK1 quantification in individual FF could constitute a new predictive biomarker of oocyte competence in addition with embryo morphokinetic parameters. TRIAL REGISTRATION NUMBER: none.

Autologous endometrial cell co-culture improves human embryo development to high-quality blastocysts: a randomized controlled trial.

RESEARCH QUESTION: Does autologous endometrial cell co-culture (AECC) improve the number of good-quality blastocysts obtained by IVF/intracytoplasmic sperm injection (ICSI), compared with conventional embryo culture medium in a broad group of patients referred to assisted reproductive technology (ART)? DESIGN: This interventional, randomized, double-blind study took place at Clinique Ovo from March 2013 to October 2015 and included 207 healthy patients undergoing an IVF or ICSI protocol, of which 71 were excluded before randomization. On the previous cycle, all participants underwent an endometrial biopsy at D5 to D7 post-ovulation, following which the endometrial cells were prepared for AECC. RESULTS: The data demonstrated that AECC significantly increased the incidence of good-quality blastocysts compared with culture in conventional media (42.6% vs 28.4%, P < 0.001). No significant differences were found in pregnancy and live birth rates. CONCLUSION: This study demonstrated the benefits of AECC on blastocyst quality compared with conventional embryo culture medium, in a broader category of patients referred to ART as opposed to other studies that concentrated on specific causes of infertility only. However, limitations of the study design should be taken into consideration; the analysis was performed using embryos rather than patients and a follow-up of children born following the treatments could not be conducted.

Thirteen years' experience of 893 PGD cycles for monogenic disorders in a publicly funded, nationally regulated regional hospital service.

This study provides an overview of preimplantation genetic diagnosis (PGD) for single gene diseases and the management of expanding indications in the context of a fully financially covered service at Montpellier's regional hospital centre. Within the framework of a restrictive law ruling PGD in France, only the parental genetic risk can be studied in embryos (concurrent aneuploidy screening is not allowed). PCR-based techniques were developed combining mutation detection and closely linked short tandem repeat markers within or flanking the affected genes, and set up more than 100 different robust fluorescent multiplex assays for 61 monogenic disorders. This strategy was used to analyse blastomeres from cleavage-stage embryos. Overall, 893 cycles were initiated in 384 couples; 727 cycles proceeded to oocyte retrieval and 608 cycles to embryo transfer, resulting in 184 deliveries. Clinical pregnancy rate per transfer, implantation and miscarriage rates were 33.6%, 25.1% and 8.8%, respectively. Our PGD programme resulted in the birth of 214 healthy babies for 162 out of 358 couples (45.3%), constituting a relevant achievement within an organizational framework that does not allow aneuploidy screening but provides equal access to PGD, both geographically and socioeconomically. This is a rare example of a fully free-of-charge PGD service.

Germline mosaicism is a pitfall in PGD for X-linked disorders. Single sperm typing detects very low frequency paternal gonadal mosaicism in a case of recurrent chondrodysplasia punctata misattributed to a maternal origin.

This manuscript presents a molecularly demonstrated gonadal mosaicism from paternal origin for X-linked dominant chondrodysplasia punctata by single sperm typing. A couple who had experienced two medical terminations of pregnancy of female fetuses was referred to our pre-implantation genetic diagnosis (PGD) centre with the diagnosis of maternally derived gonadal mosaicism. Indeed, genetic analyses of different DNA samples - including semen - from the healthy parents failed to detect the variant found in the fetuses. Six embryos, all male, were obtained during the PGD cycle. The causative variant was not detected in any embryo, whereas five embryos had inherited the 'at-risk' maternal haplotype. The assumption of a maternal gonadal mosaicism was still possible, but this finding allowed us to consider the possibility of a paternal rather than maternal gonadal mosaicism. It prompted us to perform extensive single sperm analyses, demonstrating a low-frequency paternal germline mosaicism, which led to completely different haplotype phasing and PGD counselling. In conclusion, this case further exemplifies that germline mosaicism is a pitfall in PGD where diagnosis largely relies on linkage analysis and suggests that tracing the parental inheritance through polar body analysis and/or single sperm typing experiments is of major importance for adequate genetic counselling and accurate PGD. © 2016 John Wiley & Sons, Ltd.

Apolipoprotein B is regulated by gonadotropins and constitutes a predictive biomarker of IVF outcomes.

BACKGROUND: Follicular fluid (FF) is an important micro-environment influencing oocyte growth, its development competence, and embryo viability. The FF content analysis allows to identify new relevant biomarkers, which could be predictive of in vitro fertilization (IVF) outcomes. Inside ovarian follicle, the amount of FF components from granulosa cells (GC) secretion, could be regulated by gonadotropins, which play a major role in follicle development. METHODS: This prospective study included 61 female undergoing IVF or Intra-cytoplasmic sperm injection (ICSI) procedure. Apolipoprotein B (APOB) concentrations in follicular fluid and APOB gene and protein expression in granulosa cells from reproductively aged women undergoing an in vitro fertilization program were measured. The statistical analyses were performed according to a quartile model based on the amount of APOB level found in FF. RESULTS: Amounts of APOB were detected in human FF samples (mean ± SD: 244.6 ± 185.9 ng/ml). The odds of obtaining an oocyte in the follicle and a fertilized oocyte increased significantly when APOB level in FF was higher than 112 ng/ml [i.e., including in Quartile Q 2, Q3 and Q4] (p = 0.001; p < 0.001, respectively). The probabilities of obtaining an embryo and a top quality embryo on day 2, were significantly higher if APOB levels were within the ranges of 112 and 330 ng/ml (i.e. in Q2 and Q3) or 112 and 230 ng/ml (i.e. in Q2), respectively (p < 0.001; p = 0.047, respectively). In addition, our experiments in vitro indicated that APOB gene and protein expression, along with APOB content into culture were significantly under-expressed in GC upon stimulation with gonadotropins (follicular stimulating hormone: FSH and/or human chorionic gonadotropin: hCG). CONCLUSION: We are reporting a positive and statistically significant associations between APOB and oocyte retrieval, oocyte fertilization, and embryo quality. Using an experimental study component, the authors report significant reduced APOB expression and content for luteinized granulosa cells cultured in the presence of gonadotropins.

Number of Intermediate Follicles. An Independent Risk Factor of Multiple Pregnancies in Intrauterine Insemination Cycles with Recombinant Follicle-Stimulating Hormone.

OBJECTIVE: To identify risk factors for multiple pregnancies in intrauterine insemination (IUI) cycles with recombinant follicle-stimulating hormone (r-FSH). STUDY DESIGN: A retrospective study including 205 IUI cycles with r-FSH which led to clinical pregnancies was conducted. A total of 145, singleton pregnancies and 60 multiple pregnancies were compared according to clinical characteristics and parameters of ovarian stimulation and IUI procedure. The relationships between size and number of follicles and serum estradiol (E2) levels and the risk of multiple pregnancies were investigated using multiple logistic regression analyses. RESULTS: The means of infertility length, serum E2 levels, the number of follicles 10 mm, 12 mm, and ≥ 16 mm, and the number of intermediate follicles (from 12 to ≤ 15 mm) at the day of ovulation triggering were significantly higher in the multiple pregnancy group as compared to in the singleton pregnancy group (p < 0.05). We first demonstrated that high E2 levels (≥ 1,000 pg/nL) and the number of intermediate follicles represent 2 independent and significant risk factors for multiple gestation in IUI cycles that used ovarian stimulation by r-FSH (p = 0.002 and p = 0.007, respectively). CONCLUSION: This study shows that high E2 levels and the number of intermediate follicles, independently of large follicles, can predict an increased risk of multiple pregnancy in r-FSH IUI cycles.

Birth Outcomes in DES Children and Grandchildren: A Multigenerational National Cohort Study on Informative Families.

OBJECTIVE: Diethylstilbestrol (DES), a potent synthetic nonsteroidal estrogen belonging to the family of endocrine disrupting chemicals (EDCs), can cross the placenta and may cause permanent adverse health effects in the exposed mothers, their children (exposed in utero), and also their grandchildren through germline contribution to the zygote. This study evaluated pregnancy duration and birthweight (BW) variations in the children and grandchildren born before, during, and after maternal DES treatment in the same informative families, to rule out genetic, endocrine, and environmental factors. DESIGN AND SETTING: Nationwide retrospective observational study on 529 families of DES-treated women registered at the HHORAGES-France Association. The inclusion criteria were: (i) women with at least three pregnancies and three viable children among whom the first was not exposed in utero to DES, followed by one or more children with fetal exposure to DES, and then by one or more children born after DES treatment; (ii) women with at least one pre-DES or post-DES grandchild and one DES grandchild; (iii) confirmed data on total DES dose. Women with severe pathologies or whose illness status, habitat, lifestyle habits, profession, treatment changed between pregnancies, and all mothers who reported pregnancy-related problems, were excluded. RESULTS: In all, 74 women met all criteria. The preterm birth (PTB) rate was 2.7% in pre-DES, 14.9% in DES, and 10.8% in post-DES children (Cochran-Armitage test for trend, p = 0.0095). The mean BW was higher in DES than pre-DES full-term neonates (≥37 weeks of gestation) (p = 0.007). In grandchildren, BW was not different, whereas the PTB and low BW rates were slightly increased in children of DES women. CONCLUSIONS: These data within the same informative families show the DES impact on BW and PTB in DES and post-DES children and grandchildren. In particular, mean BW was higher in DES than pre-DES full-term neonates. This result may be in opposition to previous data from American cohorts, which reported lower BW in DES children, but is consistent with animal study. Our retrospective observational study highlights a multigenerational and likely transgenerational effect of this EDC in humans.

A real-world study of ART in France (REOLA) comparing a biosimilar rFSH against the originator according to rFSH starting dose.

BACKGROUND: Since the first launch of a biosimilar recombinant follicle stimulating hormone (rFSH), Bemfola®, in Europe in 2014, it has been possible to study in routine clinical care throughout France the effectiveness of a biosimilar rFSH including according to different rFSH starting doses. METHODS: REOLA was a non-interventional, retrospective, real world study using anonymized data from 17 Assisted Reproductive Technology (ART) centres' data management systems across France including 2,319 ART ovarian stimulation cycles with Bemfola® and 4,287 ART ovarian stimulation cycles with Gonal-f®. For both products, four populations were studied according to starting dose of rFSH: < 150 IU, 150 - 224 IU, 225 - 299 IU and ≥ 300 IU. The primary endpoint was the cumulative live birth rate (cLBR) per commenced ART ovarian stimulation cycle including all subsequent fresh and frozen-thawed embryo transfers starting during a follow up period of at least 1 year following oocyte retrieval. RESULTS: A direct relationship of increasing rFSH starting dose with increasing age, increasing basal FSH, decreasing AMH and increasing body mass index was noted. No clinically relevant differences were seen in all outcomes reported, including the cLBR, between Bemfola® and Gonal-f®, but for both drugs, an association was seen with increasing rFSH starting dose and decreasing cLBR. CONCLUSIONS: The REOLA study demonstrates that the cLBR with Bemfola® is very similar to Gonal-f® across all patient subpopulations. The cLBR is inversely related to the rFSH starting dose irrespective of the drug used, and the REOLA study provides reassurance of the clinical effectiveness of a biosimilar rFSH used in a real world setting.

[The impact of oocyte cryopreservation time in oocyte donation on the clinical success rate]. / Impact de la durée de cryoconservation ovocytaire dans le cadre du don d'ovocytes sur le taux de succès clinique.

OBJECTIVES: To evaluate the impact of the cryopreservation time of vitrified oocytes on the success rates in oocyte donation cycles. METHODS: A retrospective study was carried out on 156 cycles with donated oocytes from January 2012 to September 2021. All the cycles were sorted according to the storage time of the oocytes (25 in the group 1:<3 months, 32 in the group 2: between 3 and 6 months, 39 in the group 3: between 6 and 12 months, 38 in the group 4: between 12 and 24 months and 22 in the group 5:>24 months). Clinical outcomes after ART, survival rates at thawing and oocyte fertilization rates were compared between the different cohorts stratified according to oocyte storage duration. A binary multivariate logistic regression was performed adjusting for the identified confounders. RESULTS: Prolonged storage time of vitrified oocytes had an effect on their survival post-thawing rates, but no significant effect was identified on fertilization rates or clinical outcomes. After adjusting for the confounders, the relationships between clinical outcomes and oocytes storage time did not reach statistical significance. Our study was characterized by a limited cohort with data from a single ART center. CONCLUSIONS: Our study doesn't highlight any significant difference in the use of long-stored vitrified oocytes (more than 2 years) on clinical issues in ART. The conclusion of our study needs to be verified in further studies with larger cohorts.

Differences in transcriptomic profiles of human cumulus cells isolated from oocytes at GV, MI and MII stages after in vivo and in vitro oocyte maturation.

BACKGROUND: Oocyte maturation and competence to development depends on its close relationship with cumulus cells (CCs). However, the maturation conditions of human cumulus-oocyte complexes (COCs) might affect gene expression in both oocyte and CCs. We thus compared the transcriptome profiles of CCs isolated from in vivo and in vitro matured COCs at different nuclear maturation stages. METHODS: Three groups of CCs from patients who underwent ICSI were included: CCs of patients with polycystic ovary syndrome (PCOS) referred for in vitro maturation (IVM), CCs from patients with PCOS for in vivo maturation (used as controls) and CCs from normal responders referred for in vivo maturation. CCs were isolated from COCs at the germinal vesicle, metaphase I and metaphase II stages. Microarray technology was used to analyse the global gene expression and significance analysis of microarray to compare the expression profiles of CCs from COCs at different nuclear maturation stages following IVM or in vivo maturation. Selected genes were validated by RT-qPCR. RESULTS: In CCs isolated after IVM, genes related to cumulus expansion and oocyte maturation, such as EREG, AREG and PTX3, were down-regulated, while cell cycle-related genes were up-regulated in comparison with CCs from in vivo matured COCs from PCOS and normal responder patients. Moreover, irrespective of the stage of oocyte maturation, genes involved in DNA replication, recombination and repair were up-regulated in CCs after IVM. CONCLUSIONS: The CC transcriptomic signature varies according to both the oocyte maturation stage and the maturation conditions. Our findings suggest a delay in the acquisition of the mature CC phenotype following IVM, opening a new perspective for the improvement in IVM conditions.

Brief report: benchmarking human pluripotent stem cell markers during differentiation into the three germ layers unveils a striking heterogeneity: all markers are not equal.

Pluripotent stem cells (PSC) are functionally characterized by their capacity to differentiate into all the cell types from the three germ layers. A wide range of markers, the expression of which is associated with pluripotency, has been used as surrogate evidence of PSC pluripotency, but their respective relevance is poorly documented. Here, we compared by polychromatic flow cytometry the kinetics of loss of expression of eight widely used pluripotency markers (SSEA3, SSEA4, TRA-1-60, TRA-1-81, CD24, OCT4, NANOG, and alkaline phosphatase [AP]) at days 0, 5, 7, and 9 after induction of PSC differentiation into cells representative of the three germ layers. Strikingly, each marker showed a different and specific kinetics of disappearance that was similar in all the PSC lines used and for all the induced differentiation pathways. OCT4, SSEA3, and TRA-1-60 were repeatedly the first markers to be downregulated, and their expression was completely lost at day 9. By contrast, AP activity, CD24, and NANOG proteins were still detectable at day 9. In addition, we show that differentiation markers are coexpressed with pluripotency markers before the latter begin to disappear. These results suggest that OCT4, SSEA3, and TRA-1-60 might be better to trace in vitro the emergence of pluripotent cells during reprogramming.

Insights into human endometrial receptivity from transcriptomic and proteomic data.

The appreciation of endometrial receptivity is a crucial step in assisted reproductive technology as implantation failures are thought to result, in large part, from abnormal endometrial receptivity. Using emerging omics technologies, investigators have begun to define both molecular signatures and specific biomarkers of receptive endometrium. The aim of this review was to analyse the new perspectives brought to the appreciation of endometrial receptivity by transcriptomic and proteomic technologies, involving the analysis of gene- or protein-expression-profile shifts between the pre-receptive and receptive secretory stages and how they might lead to new strategies for endometrial receptivity assessments. The use of omics as molecular tools to determine the effects of stimulation protocols on endometrial gene expression and clinical outcomes has also been investigated.