Deciphering the role of PGRMC2 in the human endometrium during the menstrual cycle and in vitro decidualization using an in vitro approach.

STUDY QUESTION: What is the human endometrial non-classical progesterone receptor (PGR) membrane component 2 (PGRMC2) expression pattern throughout the menstrual cycle and what role does it play during decidualization? SUMMARY ANSWER: Endometrial PGRMC2 expression fluctuates during the human menstrual cycle and is abundantly expressed in human endometrial stromal cells (hEnSCs) during in vitro decidualization, process where PGRMC2 is involved in embryo implantation-related pathways. WHAT IS KNOWN ALREADY: The endometrial response to progesterone is mediated by the classical and non-classical PGRs. We previously demonstrated that PGR membrane component 1 (PGRMC1) is critical for endometrial function, embryo implantation, and future placentation, however, the role(s) of PGRMC2, which is structurally similar to PGRMC1, have not been studied in the human endometrium. STUDY DESIGN, SIZE, DURATION: This prospective study comprehensively evaluated the endometrial expression of PGRMC2 throughout the human menstrual cycle and during in vitro decidualization of hEnSCs (isolated from 77 endometrial biopsies that were collected from 66 oocyte donors), using immunohistochemistry, RT-qPCR, western blot, transcriptomic, and proteomic analyses. In addition, functional analysis was carried out to validate the implication of PGRMC2 in hEnSCs during embryo invasion using an in vitro outgrowth model. PARTICIPANTS/MATERIALS, SETTING, METHODS: In vitro decidualization of hEnSCs was induced using co-treatment with cAMP and medroxyprogesterone 17-acetate progestin, and evaluated by measuring prolactin by ELISA and F-actin immunostaining. RT-qPCR was employed to compare expression with other PGRs. To reveal the function of PGRMC2 during the decidualization process, we specifically knocked down PGRMC2 with siRNAs and performed RNA-seq and quantitative proteomics techniques (SWATH-MS). The common differentially expressed genes (DEGs) and proteins (DEPs) were considered for downstream functional enrichment analysis. Finally, to verify its implication in the trophoblast invasion, an outgrowth model was carried out where hEnSCs with silenced PGRMC2 were co-cultured with human trophoblastic spheroids (JEG-3) following in vitro decidualization. MAIN RESULTS AND THE ROLE OF CHANCE: In contrast to PGRMC1 and classical PGRs, endometrial PGRMC2 gene expression was significantly lower during the late- versus mid-secretory phase (P < 0.05). Accordingly, the elevated PGRMC2 protein abundance observed in the endometrial epithelial glands throughout the menstrual cycle dropped in the late secretory phase, when abundance decreased in all endometrial compartments. Nevertheless, PGRMC2 protein increased during the mid-secretory phase in stromal and glandular cells, and PGRMC2 mRNA (P < 0.0001) and protein (P < 0.001) levels were significantly enhanced in the membranes/organelles of decidualized hEnSCs, compared to non-decidualized hEnSCs. Notably, PGRMC1 and PGRMC2 mRNA were significantly more abundant than classical PGRs throughout menstrual cycle phases and in decidualized and non-decidualized hEnSCs (P < 0.05). RNA-seq and proteomics data revealed 4687 DEGs and 28 DEPs, respectively, in decidualized hEnSCs after PGRMC2 silencing. While functional enrichment analysis showed that the 2420 upregulated genes were mainly associated with endoplasmic reticulum function, vesicular transport, morphogenesis, angiogenesis, cell migration, and cell adhesion, the 2267 downregulated genes were associated with aerobic respiration and protein biosynthesis. The protein enrichment analysis showed that 4 upregulated and 24 downregulated proteins were related to aerobic respiration, cellular response, metabolism, localization of endoplasmic reticulum proteins, and ribonucleoside biosynthesis routes. Finally, PGRMC2 knockdown significantly compromised the ability of the decidualized hEnSCs to support trophoblast expansion in an outgrowth model (P < 0.05). LARGE-SCALE DATA: Transcriptomic data are available via NCBI's Gene Expression Omnibus (GEO) under GEO Series accession number GSE251843 and proteomic data via ProteomeXchange with identifier PXD048494. LIMITATIONS, REASONS FOR CAUTION: The functional analyses were limited by the discrete number of human endometrial biopsies. A larger sample size is required to further investigate the potential role(s) of PGRMC2 during embryo implantation and maintenance of pregnancy. Further, the results obtained in the present work should be taken with caution, as the use of a pure primary endometrial stromal population differentiated in vitro does not fully represent the heterogeneity of the endometrium in vivo, nor the paracrine communications occurring between the distinct endometrial cell types. WIDER IMPLICATIONS OF THE FINDINGS: The repression of endometrial PGRMC2 during the late- versus mid-secretory phase, together with its overexpression during decidualization and multiple implications with embryo implantation not only highlighted the unknown roles of PGRMC2 in female reproduction but also the potential to exploit PGRMC2 signaling pathways to improve assisted reproduction treatments in the future. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by Instituto de Salud Carlos III (ISCIII) granted to F.D. (PI20/00405 and PI23/00860), co-funded by the European Union. Y.M.-L. was supported by a predoctoral research grant from Generalitat Valenciana (ACIF/2019/262). R.G.-M. was supported by Generalitat Valenciana (CIAPOT/2022/15). P.d.C. was supported by a predoctoral grant for training in research into health (PFIS FI20/00086) from the Instituto de Salud Carlos III. I.D.-H. was supported by the Spanish Ministry of Science, Innovation and Universities (FPU18/01550). A.P. was supported by the Instituto de Salud Carlos III (PFIS FI18/00009). This research was also supported by IVI Foundation-RMA Global (1911-FIVI-103-FD). The authors declare no conflict of interest.

Serum progesterone is lower in ovarian stimulation with highly purified HMG compared to recombinant FSH owing to a different regulation of follicular steroidogenesis: a randomized controlled trial.

STUDY QUESTION: Does ovarian stimulation with highly purified (hp)-HMG protect from elevated progesterone in the follicular phase compared to recombinant FSH (r-FSH) cycles through a different regulation of follicular steroidogenesis? SUMMARY ANSWER: hp-HMG enhanced the Δ4 pathway from pregnenolone to androstenodione leading to lower serum progesterone at the end of the cycle, while r-FSH promoted the conversion of pregnenolone to progesterone causing higher follicular phase progesterone levels. WHAT IS KNOWN ALREADY: Elevated progesterone in the follicular phase has been related to lower clinical outcome in fresh IVF cycles. Progesterone levels are positively correlated to ovarian response, and some studies have shown that when r-FSH alone is used for ovarian stimulation serum progesterone levels on the day of triggering are higher than when hp-HMG is given. Whether this is caused by a lower ovarian response in hp-HMG cycles or to a difference in follicular steroidogenesis in the two ovarian stimulation regimens has not been well characterized. STUDY DESIGN, SIZE, DURATION: A randomized controlled trial including 112 oocyte donors undergoing ovarian stimulation with GnRH antagonists and 225 IU/day of r-FSH (n = 56) or hp-HMG (n = 56) was carried out in a university-affiliated private infertility clinic. Subjects were recruited between October 2016 and June 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: The women were aged 18-35 years with a regular menstrual cycle (25-35 days) and normal ovarian reserve (serum anti-Müllerian hormone (AMH) = 10-30 pMol/l) undergoing ovarian stimulation for oocyte donation. FSH, LH, estradiol (E2), estrone, progesterone, pregnenolone, 17-OH-progesterone, androstenodione, dehidroepiandrostenodione, and testosterone were determined on stimulation Days 1, 4, 6, and 8 and on day of triggering in serum and in follicular fluid. Samples were frozen at -20°C until assay. Total exposures across the follicular phase were compared by polynomic extrapolation. MAIN RESULTS AND THE ROLE OF CHANCE: Subjects in both groups were comparable in terms of age, BMI, and AMH levels. Ovarian response was also similar: 17.5 ± 7.9 (mean ± SD) versus 16.5 ± 7.5 oocytes with r-FSH and hp-HMG, respectively (P = 0.49). Serum progesterone (ng/ml) on day of trigger was 0.46 ± 0.27 in the hp-HMG group versus 0.68 ± 0.50 in the r-FSH group (P = 0.010). Differences for progesterone were also significant on stimulation days 6 and 8. The pregnenolone: progesterone ratio was significantly increased in the r-FSH group from stimulation day 8 to the day of trigger (P = 0.019). Serum androstenodione (ng/ml) on day of trigger was 3.0 ± 1.4 in the hp-HMG group versus 2.4 ± 1.1 in the r-FSH group (P = 0.015). Differences in adrostenodione were also significant on stimulation Day 8. The pregnenolone:androstenodione ratio was significantly higher in the hp-HMG group (P = 0.012) on Days 6 and 8 and trigger. There were no other significant differences between groups. Follicular fluid E2, FSH, LH, dehidroepioandrostenodione, androstenodione, and testosterone were significantly higher in the hp-HMG than r-FSH group. No differences were observed for progesterone, estrone, 17-OH-progesterone, and pregnenolone in follicular fluid. LIMITATIONS, REASONS FOR CAUTION: All women included in the study were young, not infertile, and had a normal BMI and a good ovarian reserve. The findings might be different in other patient subpopulations. Hormone analyses with immunoassays are subject to intra-assay variations that may influence the results. WIDER IMPLICATIONS OF THE FINDINGS: Stimulation with hp-HMG may prevent progesterone elevation at the end of the follicular phase because of a different follicular steroidogenesis pathway, regardless of ovarian response. This should be considered, particularly in patients at risk of having high progesterone levels at the end of the follicular phase when a fresh embryo transfer is planned. STUDY FUNDING/COMPETING INTEREST(S): Roche Diagnostics provided unrestricted funding for all serum and follicular fluid hormone determinations. J.L.R., M.M., and A.P. have nothing to declare. E.B. has received consulting fees from Ferring, Merck, Gedeon Richter, and Roche and has participated in a research cooperation with Gedeon-Richter. In addition, the author has participated in speakers' bureau and received fees from Ferring, Gedeon Richter, Merck, and Roche. P.A. has received consulting fees from MSD and has participated in speakers' bureau and received fees from Ferring. P.A. also declares travel/meeting support from MSD. E.L. has received consulting fees from Ferring and MSD. In addition, the author has participated in a research cooperation with Gedeon-Richter. Also, the author has participated in speakers' bureau and received fees from Ferring and IBSA, as well as travel/meeting support from IBSA and Gedeon Richter. E.B., P.A., and E.L. also own stocks in IVIRMA Valencia. TRIAL REGISTRATION NUMBER: NCT: NCT02738580. TRIAL REGISTER DATE: 19 February 2016. DATE OF FIRST PATIENT'S ENROLMENT: 03 October 2016.

Antioxidant Supplementation Alleviates Mercury-Induced Cytotoxicity and Restores the Implantation-Related Functions of Primary Human Endometrial Cells.

Mercury (Hg) cytotoxicity, which is largely mediated through oxidative stress (OS), can be relieved with antioxidants. Thus, we aimed to study the effects of Hg alone or in combination with 5 nM N-Acetyl-L-cysteine (NAC) on the primary endometrial cells' viability and function. Primary human endometrial epithelial cells (hEnEC) and stromal cells (hEnSC) were isolated from 44 endometrial biopsies obtained from healthy donors. The viability of treated endometrial and JEG-3 trophoblast cells was evaluated via tetrazolium salt metabolism. Cell death and DNA integrity were quantified following annexin V and TUNEL staining, while the reactive oxygen species (ROS) levels were quantified following DCFDA staining. Decidualization was assessed through secreted prolactin and the insulin-like growth factor-binding protein 1 (IGFBP1) in cultured media. JEG-3 spheroids were co-cultured with the hEnEC and decidual hEnSC to assess trophoblast adhesion and outgrowth on the decidual stroma, respectively. Hg compromised cell viability and amplified ROS production in trophoblast and endometrial cells and exacerbated cell death and DNA damage in trophoblast cells, impairing trophoblast adhesion and outgrowth. NAC supplementation significantly restored cell viability, trophoblast adhesion, and outgrowth. As these effects were accompanied by the significant decline in ROS production, our findings originally describe how implantation-related endometrial cell functions are restored in Hg-treated primary human endometrial co-cultures by antioxidant supplementation.

Establishment of Adenomyosis Organoids as a Preclinical Model to Study Infertility.

Adenomyosis is related to infertility and miscarriages, but so far there are no robust in vitro models that reproduce its pathological features to study the molecular mechanisms involved in this disease. Endometrial organoids are in vitro 3D models that recapitulate the native microenvironment and reproduce tissue characteristics that would allow the study of adenomyosis pathogenesis and related infertility disorders. In our study, human endometrial biopsies from adenomyosis (n = 6) and healthy women (n = 6) were recruited. Organoids were established and hormonally differentiated to recapitulate midsecretory and gestational endometrial phases. Physiological and pathological characteristics were evaluated by immunohistochemistry, immunofluorescence, qRT-PCR, and ELISA. Secretory and gestational organoids recapitulated in vivo glandular epithelial phenotype (pan-cytokeratin, Muc-1, PAS, Laminin, and Ki67) and secretory and gestational features (α-tubulin, SOX9, SPP1, PAEP, LIF, and 17ßHSD2 expression and SPP1 secretion). Adenomyosis organoids showed higher expression of TGF-ß2 and SMAD3 and increased gene expression of SPP1, PAEP, LIF, and 17ßHSD2 compared with control organoids. Our results demonstrate that organoids derived from endometria of adenomyosis patients and differentiated to secretory and gestational phases recapitulate native endometrial-tissue-specific features and disease-specific traits. Adenomyosis-derived organoids are a promising in vitro preclinical model to study impaired implantation and pregnancy disorders in adenomyosis and enable personalized drug screening.

Medroxyprogesterone acetate is a useful alternative to a gonadotropin-releasing hormone antagonist in oocyte donation: a randomized, controlled trial.

OBJECTIVE: To compare ovarian response and reproductive outcomes in oocyte donors undergoing pituitary suppression with medroxyprogesterone acetate (MPA) versus those undergoing conventional treatment with a gonadotropin-releasing hormone (GnRH) antagonist. DESIGN: A prospective, randomized, controlled trial of cycles was conducted from October 2017 to June 2019 to evaluate ovarian response in terms of the number of oocytes. The reproductive outcomes of the recipients were retrospectively analyzed later. SETTING: A university-affiliated private in vitro fertilization center. PATIENT(S): We randomly divided 318 donors into 2 groups in a 1:1 ratio. The oocytes obtained were assigned to 364 recipients. One hundred sixty-one donors were treated with a daily dose of 10 mg of MPA administered orally from the beginning of ovarian stimulation (OS), and 156 were treated with a GnRH antagonist (initiated once the leading follicle reached a diameter of 13 mm). Transvaginal ultrasound was performed, and serum estradiol, luteinizing hormone, and progesterone levels were recorded during monitoring. The following additional parameters were analyzed: endocrine profile (in follicular fluid), number of metaphase II oocytes, and pregnancy outcome. INTERVENTION(S): The donors included in the study group were stimulated using recombinant follicle-stimulating hormone and MPA at 10 mg/day, simultaneously begun on cycle day 2 or 3. Ovulation was induced using a GnRH agonist when dominant follicles matured. A short protocol with ganirelix at 0.25 mg/day was used for the control group. Oocytes were assigned to the recipients, followed by routine in vitro fertilization procedures in which 1 embryo was usually transferred. MAIN OUTCOME MEASURE(S): The primary outcome measure was the numbers of oocytes and metaphase II oocytes retrieved. The secondary outcomes were the incidence of premature luteinizing hormone surge, serum and follicular fluid hormone profiles, and clinical pregnancy outcomes in the recipient group. RESULT(S): The number of oocytes retrieved was 21.4 ± 11.7 in the MPA group and 21.2 ± 9.2 in the antagonist group (mean difference 0.14; 95% confidence interval -2.233, 2.517). The total dose of recombinant follicle-stimulating hormone, duration of OS, and endocrine profiles of the serum and follicular fluids were comparable in the 2 groups. No early ovulation was observed in either group. No statistically significant differences with respect to implantation rate (68.1% in the MPA group vs. 62% in the antagonist group), clinical pregnancy rate (64.5% in the MPA group vs. 57.8 in the antagonist group), ongoing pregnancy rate (55.4% in the MPA group vs. 48.5% in the antagonist group), live birth rate (55.1% in the MPA group vs. 48.5% in the antagonist group), or cumulative live birth rate (73.8% in the MPA group vs. 70.7% in the antagonist group) were observed between the groups. CONCLUSION(S): The administration of MPA resulted in oocyte retrieval rates, endocrine profiles, viable embryo numbers, and pregnancy outcomes similar to those achieved with the GnRH antagonist. Therefore, MPA can be recommended for OS in oocyte donation because it permits a more patient-friendly approach. CLINICAL TRIAL REGISTRATION NUMBER: NCT03300960.

Impact of low serum progesterone levels on the day of embryo transfer on pregnancy outcome: a prospective cohort study in artificial cycles with vaginal progesterone.

STUDY QUESTION: Is there a serum progesterone (P) threshold on the day of embryo transfer (ET) in artificial endometrium preparation cycles below which the chances of ongoing pregnancy are reduced? SUMMARY ANSWER: Serum P levels <8.8 ng/ml on the day of ET lower ongoing pregnancy rate (OPR) in both own or donated oocyte cycles. WHAT IS KNOWN ALREADY: We previously found that serum P levels <9.2 ng/ml on the day of ET significantly decrease OPR in a sample of 211 oocyte donation recipients. Here, we assessed whether these results are applicable to all infertile patients under an artificial endometrial preparation cycle, regardless of the oocyte origin. STUDY DESIGN, SIZE, DURATION: This prospective cohort study was performed between September 2017 and November 2018 and enrolled 1205 patients scheduled for ET after an artificial endometrial preparation cycle with estradiol valerate and micronized vaginal P (MVP, 400 mg twice daily). PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients ≤50 years old with a triple-layer endometrium ≥6.5 mm underwent transfer of one or two blastocysts. A total of 1150 patients treated with own oocytes without preimplantation genetic testing for aneuploidies (PGT-A) (n = 184), own oocytes with PGT-A (n = 308) or donated oocytes (n = 658) were analyzed. The primary endpoint was the OPR beyond pregnancy week 12 based on serum P levels measured immediately before ET. MAIN RESULTS AND THE ROLE OF CHANCE: Women with serum P levels <8.8 ng/ml (30th percentile) had a significantly lower OPR (36.6% vs 54.4%) and live birth rate (35.5% vs 52.0%) than the rest of the patients. Multivariate logistic regression showed that serum P < 8.8 ng/ml was an independent factor influencing OPR in the overall population and in the three treatment groups. A significant negative correlation was observed between serum P levels and BMI, weight and time between the last P dose and blood tests and a positive correlation was found with age, height and number of days on HRT. Multivariate logistic regression showed that only body weight was an independent factor for presenting serum P levels <8.8 ng/ml. Obstetrical and perinatal outcomes did not differ in patients with ongoing pregnancy regardless of serum P levels being above/below 8.8 ng/ml. LIMITATIONS, REASONS FOR CAUTION: Only women with MVP were included. Extrapolation to other P administration forms needs to be validated. WIDER IMPLICATIONS OF THE FINDINGS: This study identified the threshold of serum P as 8.8 ng/ml on the day of ET for artificial endometrial preparation cycles necessary to optimize outcomes, in cycles with own or donated oocytes. One-third of patients receiving MVP show inadequate levels of serum P that, in turn, impact the success of the ART cycle. Monitoring P levels in the mid-luteal phase is recommended when using MVP to adjust the doses according to the needs of the patient. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: NCT03272412.

Single-cell transcriptomic atlas of the human endometrium during the menstrual cycle.

In a human menstrual cycle the endometrium undergoes remodeling, shedding and regeneration, all of which are driven by substantial gene expression changes in the underlying cellular hierarchy. Despite its importance in human fertility and regenerative biology, our understanding of this unique type of tissue homeostasis remains rudimentary. We characterized the transcriptomic transformation of human endometrium at single-cell resolution across the menstrual cycle, resolving cellular heterogeneity in multiple dimensions. We profiled the behavior of seven endometrial cell types, including a previously uncharacterized ciliated cell type, during four major phases of endometrial transformation, and found characteristic signatures for each cell type and phase. We discovered that the human window of implantation opens with an abrupt and discontinuous transcriptomic activation in the epithelia, accompanied with a widespread decidualization feature in the stromal fibroblasts. Our study provides a high-resolution molecular and cellular characterization of human endometrial transformation across the menstrual cycle, providing insights into this essential physiological process.

Endometrial Liquid Biopsy Provides a miRNA Roadmap of the Secretory Phase of the Human Endometrium.

CONTEXT: Endometrial liquid biopsy (ELB) is a minimally invasive alternative for research and diagnosis in endometrial biology. OBJECTIVE: We sought to establish an endometrial micro ribonucleic acid (miRNA) roadmap based on ELB during the secretory phase of the menstrual cycle in both natural and hormonal replacement therapy (HRT) cycles. DESIGN: Human ELB samples (n = 58) were obtained from healthy ovum donors undergoing a natural and an HRT cycle consecutively. miRNA profiles were identified using next-generation sequencing (NGS). For functional analysis, messenger ribonucleic acid targets were chosen among those reported in the endometrial receptivity analysis. RESULTS: The human endometrial secretory phase is characterized by a dynamic miRNA secretion pattern that varies from the prereceptive to the receptive stages. No differences in miRNA profiles were found among natural versus HRT cycles in the same women, reinforcing the similarities in functional and clinical outcomes in natural versus medicated cycles. Bioinformatic analysis revealed 62 validated interactions and 81 predicted interactions of miRNAs differentially expressed in the HRT cycle. Annotation of these genes linked them to 51 different pathways involved in endometrial receptivity. CONCLUSION: This NGS-based study describes the miRNA signature in human ELB during the secretory phase of natural and HRT cycles. A consistent endometrial miRNA signature was observed in the acquisition of endometrial receptivity. Interestingly, no significant differences in miRNA expression were found in natural versus HRT cycles reinforcing the functional clinical similarities between both approaches.

iTRAQ comparison of proteomic profiles of endometrial receptivity.

Endometrial receptivity is a limiting step in human reproduction. A disruption in the development of endometrial receptivity is responsible for recurrent implantation failures (RIF) of endometrial origin. To understand the molecular mechanisms behind the endometrial receptivity process, we used the isobaric tag for relative and absolute quantitation (iTRAQ) method to compare three different endometrial statuses: fertile women, intrauterine device (IUD) carriers, and RIF patients. Overall, iTRAQ allowed identified 1889 non-redundant proteins. Of these, 188 were differentially expressed proteins (DEP) (p-value < .05). Pairwise comparisons revealed 133 significant DEP in fertile vs. IUD carriers and 158 DEP in RIF vs. IUD carriers. However, no DEP were identified between fertile and RIF patients. Western blot validation of three DEP involved in endometrial receptivity (plastin 2, lactotransferrin, and lysozyme) confirmed our iTRAQ results. Moreover, functional KEGG enrichment revealed that complement and coagulation cascades and peroxisome were the two most significant pathways for the RIF vs. IUD comparison and ribosome and spliceosome for the fertile vs. IUD comparison, as possible important pathways involved in the endometrial receptivity acquisition. The lack of DEP between fertile and RIF patient endometria suggest that idiopathic RIF may not have an endometrial origin, with other as-yet-unknown factors involved. SIGNIFICANCE: A pilot study where a comparison of the endometrial protein profile from women with different endometrial receptive grade (fertile women, IUD carriers and RIF patients) during the same period of time (overlapping with the window of implantation) of a hormone replacement therapy was performed using a high-throughput proteomic technique. This approach lead us to better understand the molecular mechanisms undergoing endometrial receptivity, a time-limiting step to achieve pregnancy in humans. Moreover, the number of samples per group (10 Fertile women, 10 IUD carriers and 8 RIF patients) according to the methodology here employed (8plex iTRAQ), give more robustness to our results. Our findings confirm that an IUD introduces numerous changes in the endometrial protein profile when compared to fertile and RIF endometria, revealing some key proteins involved in endometrial receptivity. Finding no significant differences between Fertile and RIF patient endometria could suggest that other as-yet-unknown factors could be involved in the etiology of idiopathic RIF.

A Higher Ovarian Response after Stimulation for IVF Is Related to a Higher Number of Euploid Embryos.

This study has analysed the relationship between ovarian response and the number of euploid embryos. This is a post hoc analysis of a subset of data generated during a prospective cohort study previously published. Forty-six oocyte donors were subjected to ovarian stimulation with 150 IU of rFSH and 75 IU of hp-hMG in a GnRH agonist long protocol. Preimplantation genetic screening was performed in all viable embryos. We observed a positive relationship between ovarian response and the number of euploid embryos. When ovarian response was above the median (≥17 oocytes), the mean number of euploid embryos per donor was 5.0 ± 2.4, while when <17 oocytes were obtained the mean number of euploid embryos was 2.7 ± 1.4 (p = 0.000). Aneuploidy rate did not increase with ovarian response or gonadotropin doses. Also, the number of euploid embryos was inversely related to the amount of gonadotropins needed per oocyte obtained (ovarian sensitivity index). These results suggest that the number of euploid embryos available for embryo transfer increases as the number of oocytes obtained does. Considering the total number of euploid embryos seems more relevant than the aneuploidy rate.

Evidence that the endometrial microbiota has an effect on implantation success or failure.

BACKGROUND: Bacterial cells in the human body account for 1-3% of total body weight and are at least equal in number to human cells. Recent research has focused on understanding how the different bacterial communities in the body (eg, gut, respiratory, skin, and vaginal microbiomes) predispose to health and disease. The microbiota of the reproductive tract has been inferred from the vaginal bacterial communities, and the uterus has been classically considered a sterile cavity. However, while the vaginal microbiota has been investigated in depth, there is a paucity of consistent data regarding the existence of an endometrial microbiota and its possible impact in reproductive function. OBJECTIVE: This study sought to test the existence of an endometrial microbiota that differs from that in the vagina, assess its hormonal regulation, and analyze the impact of the endometrial microbial community on reproductive outcome in infertile patients undergoing in vitro fertilization. STUDY DESIGN: To identify the existence of an endometrial microbiota, paired samples of endometrial fluid and vaginal aspirates were obtained simultaneously from 13 fertile women in prereceptive and receptive phases within the same menstrual cycle (total samples analyzed n = 52). To investigate the hormonal regulation of the endometrial microbiota during the acquisition of endometrial receptivity, endometrial fluid was collected at prereceptive and receptive phases within the same cycle from 22 fertile women (n = 44). Finally, the reproductive impact of an altered endometrial microbiota in endometrial fluid was assessed by implantation, ongoing pregnancy, and live birth rates in 35 infertile patients undergoing in vitro fertilization (total samples n = 41) with a receptive endometrium diagnosed using the endometrial receptivity array. Genomic DNA was obtained either from endometrial fluid or vaginal aspirate and sequenced by 454 pyrosequencing of the V3-V5 region of the 16S ribosomal RNA (rRNA) gene; the resulting sequences were taxonomically assigned using QIIME. Data analysis was performed using R packages. The χ2 test, Student t test, and analysis of variance were used for statistical analyses. RESULTS: When bacterial communities from paired endometrial fluid and vaginal aspirate samples within the same subjects were interrogated, different bacterial communities were detected between the uterine cavity and the vagina of some subjects. Based on its composition, the microbiota in the endometrial fluid, comprising up to 191 operational taxonomic units, was defined as a Lactobacillus-dominated microbiota (>90% Lactobacillus spp.) or a non-Lactobacillus-dominated microbiota (<90% Lactobacillus spp. with >10% of other bacteria). Although the endometrial microbiota was not hormonally regulated during the acquisition of endometrial receptivity, the presence of a non-Lactobacillus-dominated microbiota in a receptive endometrium was associated with significant decreases in implantation [60.7% vs 23.1% (P = .02)], pregnancy [70.6% vs 33.3% (P = .03)], ongoing pregnancy [58.8% vs 13.3% (P = .02)], and live birth [58.8% vs 6.7% (P = .002)] rates. CONCLUSION: Our results demonstrate the existence of an endometrial microbiota that is highly stable during the acquisition of endometrial receptivity. However, pathological modification of its profile is associated with poor reproductive outcomes for in vitro fertilization patients. This finding adds a novel microbiological dimension to the reproductive process.

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.

The accuracy and reproducibility of the endometrial receptivity array is superior to histology as a diagnostic method for endometrial receptivity.

OBJECTIVE: To compare the accuracy and reproducibility of the endometrial receptivity array (ERA) versus standard histologic methods. DESIGN: A comparative prospective study (May 2008-May 2012). SETTING: University-affiliated infertility clinic. PATIENT(S): Eighty-six healthy oocyte donors, regularly cycling, aged 20-34 years with a body mass index (BMI) of 19-25 kg/m(2). INTERVENTION(S): Endometrial biopsies were collected throughout the menstrual cycle. For the accuracy study, 79 samples were grouped into two cohorts: the training set (n = 79) for ERA machine-learning training and dating, and a dating subset (n = 49) for comparison between histologic and ERA dating. For the reproducibility study, seven women underwent ERA testing and it was repeated in the same patients on the same day of their cycle 29-40 months later. MAIN OUTCOME MEASURE(S): Concordance of histologic and ERA dating related to LH as a reference, and interobserver variability between pathologists were statistically analyzed by the quadratic weighted Kappa index. The ERA reproducibility was tested and its gene expression visualized by principal component analysis. RESULT(S): For each pathologist, concordance against LH peak yielded values of 0.618 (0.446-0.791) and 0.685 (0.545-0.824). Interobserver variability between pathologists yielded a Kappa index of 0.622 (0.435-0.839). Concordance for ERA dating against LH peak showed a value of 0.922 (0.815-1.000). Reproducibility of the ERA test was 100% consistent. CONCLUSION(S): The ERA is more accurate than histologic dating and is a completely reproducible method for the diagnosis of endometrial dating and receptivity status.

Endometrial gene expression in the window of implantation is altered in obese women especially in association with polycystic ovary syndrome.

OBJECTIVE: To determine whether luteal phase endometrial transcriptome is altered in obese women during the window of implantation (WOI), considering the presence of infertility, fat distribution and association with polycystic ovary syndrome (PCOS). DESIGN: Prospective study. SETTING: University-affiliated infertility clinic, between May 2007 and March 2009. PATIENT(S): One control group of women with normal weight (n=4), and four study groups of obese women (n=6 each one) according to the association with infertility, PCOS, and ovarian stimulation. INTERVENTION(S): The endometrium was biopsied 7 days after LH surge or hCG administration in 28 women. MAIN OUTCOME MEASURE(S): Endometrial gene expression during the WOI. RESULT(S): One hundred and fifty-one genes were dysregulated in obese groups compared with controls. This dysregulation was more pronounced when infertility was associated. The biologic processes of these genes belonged mainly to development and regulation of different biological functions such as transcription and biosynthesis. The molecular functions overrepresented were transcription and peptide receptor activity. The endometrium of obese women with PCOS showed dysregulated genes related to biologic processes such as development, morphogenesis, and the immune system, as well as different molecular functions such as protein binding, binding, growth factor activity, and carboxylic acid transmembrane transporter activity. Some of these genes have been previously related to implantation and unexplained infertility. CONCLUSION(S): Obese women present a different endometrial gene expression than controls during the WOI, which is more pronounced when infertility or polycystic ovary syndrome are associated.

A genomic diagnostic tool for human endometrial receptivity based on the transcriptomic signature.

OBJECTIVE: To create a genomic tool composed of a customized microarray and a bioinformatic predictor for endometrial dating and to detect pathologies of endometrial origin. To define the transcriptomic signature of human endometrial receptivity. DESIGN: Two cohorts of endometrial samples along the menstrual cycle were used: one to select the genes to be included in the customized microarray (endometrial receptivity array [ERA]), and the other to be analyzed by ERA to train the predictor for endometrial dating and to define the transcriptomic signature. A third cohort including pathological endometrial samples was used to train the predictor for pathological classification. SETTING: Healthy oocyte donors and patients. PATIENT(S): Healthy fertile women (88) and women with implantation failure (5) or hydrosalpinx (2). INTERVENTION(S): Human endometrial biopsies. MAIN OUTCOME MEASURE(S): The gene expression of endometrial biopsies. RESULT(S): The ERA included 238 selected genes. The transcriptomic signature was defined by 134 genes. The predictor showed a specificity of 0.8857 and sensitivity of 0.99758 for endometrial dating, and a specificity of 0.1571 and a sensitivity of 0.995 for the pathological classification. CONCLUSION(S): This diagnostic tool can be used clinically in reproductive medicine and gynecology. The transcriptomic signature is a potential endometrial receptivity biomarkers cluster.

Prospective cohort study in high responder oocyte donors using two hormonal stimulation protocols: impact on embryo aneuploidy and development.

BACKGROUND: Ovarian stimulation regimens for in vitro fertilization seem to have a deleterious effect on oocyte quality and embryo aneuploidy in a dose-dependent manner. This study aims to test the influence of gonadotrophin doses on embryo aneuploidy rates. METHODS: A total of 32 young oocyte donors with a high response to ovarian stimulation, were included in the study. Two subsequent stimulation treatments were performed in each donor: first, a standard dose cycle using a 225 IU starting dose of recombinant FSH (r-FSH) and secondly, a reduced dose cycle with a starting dose of 150 IU r-FSH. In both cycles, GnRH agonist co-treatment was used for down-regulation. Ovarian response, embryo development and aneuploidy for chromosomes 13, 15, 16, 17, 18, 21, 22, X and Y were the main outcomes of the study. RESULTS: A total of 22 donors completed both treatments with different gonadotrophin doses. In the remaining 10 donors, the reduced dose cycle was cancelled due to low ovarian response. In those donors who completed both regimens, significant increases in rates of fertilization and chromosomally normal blastocysts were observed in the reduced dose cycle. No differences were observed in pregnancy and implantation rates in recipients who received oocytes from standard and reduced doses cycles. CONCLUSIONS: Despite the limited numbers in our study, we can conclude that in high responder donors, a decrease in the gonadotrophin dose could improve fertilization rates and embryo quality. However, due to the reduced oocyte numbers with lower doses, a similar reproductive outcome in terms of live births would be expected.