Identifying and optimizing human endometrial gene expression signatures for endometrial dating.

STUDY QUESTION: What are the key considerations for developing an enhanced transcriptomic method for secretory endometrial tissue dating? SUMMARY ANSWER: Multiple gene expression signature combinations can serve as biomarkers for endometrial dating, but their predictive performance is variable and depends on the number and identity of the genes included in the prediction model, the dataset characteristics and the technology employed for measuring gene expression. WHAT IS KNOWN ALREADY: Among the new generation of transcriptomic endometrial dating (TED) tools developed in the last decade, there exists variation in the technology used for measuring gene expression, the gene makeup and the prediction model design. A detailed study, comparing prediction performance across signatures for understanding signature behaviour and discrepancies in gene content between them, is lacking. STUDY DESIGN, SIZE, DURATION: A multicentre prospective study was performed between July 2018 and October 2020 at five different centres from the same group of clinics (Spain). This study recruited 281 patients and finally included in the gene expression analysis 225 Caucasian patients who underwent IVF treatment. After preprocessing and batch effect filtering, gene expression measurements from 217 patients were combined with artificial intelligence algorithms (support vector machine, random forest and k-nearest neighbours) allowing evaluation of different prediction models. In addition, secretory-phase endometrial transcriptomes from gene expression omnibus (GEO) datasets were analysed for 137 women, to study the endometrial dating capacity of genes independently and grouped by signatures. This provided data on the consistency of prediction across different gene expression technologies and datasets. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial biopsies were analysed using a targeted TruSeq (Illumina) custom RNA expression panel called the endometrial dating panel (ED panel). This panel included 301 genes previously considered relevant for endometrial dating as well as new genes selected for their anticipated value in detecting the secretory phase. Final samples (n = 217) were divided into a training set for signature discovery and an independent testing set for evaluation of predictive performance of the new signature. In addition, secretory-phase endometrial transcriptomes from GEO were analysed for 137 women to study endometrial dating capacity of genes independently and grouped by signatures. Predictive performance among these signatures was compared according to signature gene set size. MAIN RESULTS AND THE ROLE OF CHANCE: Testing of the ED panel allowed development of a model based on a new signature of 73 genes, which we termed 'TED' and delivers an enhanced tool for the consistent dating of the secretory phase progression, especially during the mid-secretory endometrium (3-8 days after progesterone (P) administration (P + 3-P + 8) in a hormone replacement therapy cycle). This new model showed the best predictive capacity in an independent test set for staging the endometrial tissue in the secretory phase, especially in the expected window of implantation (average of 114.5 ± 7.2 h of progesterone administered; range in our patient population of 82-172 h). Published sets of genes, in current use for endometrial dating and the new TED genes, were evaluated in parallel in whole-transcriptome datasets and in the ED panel dataset. TED signature performance was consistently excellent for all datasets assessed, frequently outperforming previously published sets of genes with a smaller number of genes for dating the endometrium in the secretory phase. Thus, this optimized set exhibited prediction consistency across datasets. LARGE SCALE DATA: The data used in this study is partially available at GEO database. GEO identifiers GSE4888, GSE29981, GSE58144, GSE98386. LIMITATIONS, REASONS FOR CAUTION: Although dating the endometrial biopsy is crucial for investigating endometrial progression and the receptivity process, further studies are needed to confirm whether or not endometrial dating methods in general are clinically useful and to guide the specific use of TED in the clinical setting. WIDER IMPLICATIONS OF THE FINDINGS: Multiple gene signature combinations provide adequate endometrial dating, but their predictive performance depends on the identity of the genes included, the gene expression platform, the algorithms used and dataset characteristics. TED is a next-generation endometrial assessment tool based on gene expression for accurate endometrial progression dating especially during the mid-secretory. STUDY FUNDING/COMPETING INTEREST(S): Research funded by IVI Foundation (1810-FIVI-066-PD). P.D.-G. visiting scientist fellowship at Oxford University (BEFPI/2010/032) and Josefa Maria Sanchez-Reyes' predoctoral fellowship (ACIF/2018/072) were supported by a program from the Generalitat Valenciana funded by the Spanish government. A.D.-P. is supported by the FPU/15/01398 predoctoral fellowship from the Ministry of Science, Innovation and Universities (Spanish Government). D.W. received support from the NIHR Oxford Biomedical Research Centre. The authors do not have any competing interests to declare.

Analysis of serum and endometrial progesterone in determining endometrial receptivity.

STUDY QUESTION: Is there a relationship between serum and endometrial progesterone (P4) levels, including P4 and metabolites (oestrone, oestradiol and 17α-hydroxyprogesterone), and endometrial receptivity? SUMMARY ANSWER: Serum P4 levels were not correlated with endometrial P4, nor associated with endometrial receptivity as determined by the ERA® test; however, endometrial P4 and 17α-hydroxyprogesterone levels were positively correlated and related to endometrial receptivity by ERA. WHAT IS KNOWN ALREADY: Acquisition of endometrial receptivity is governed by P4, which induces secretory transformation. A close relationship between serum P4 and pregnancy outcome is reported for hormone replacement therapy (HRT) cycles. However, the relationship between serum and uterine P4 levels has not been described, and it is unknown whether uterine receptivity depends more on serum or uterine P4 levels. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was performed during March 2018-2019 in 85 IVF patients undergoing an evaluation-only HRT cycle with oestradiol valerate (6 mg/day) and micronised vaginal progesterone (400 mg/12 h). PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were under 50 years of age, had undergone at least one failed IVF cycle, had no uterine pathology, and had adequate endometrial thickness (> 6.5 mm). The study was conducted at IVI Valencia and IVI Foundation. An endometrial biopsy and a blood sample were collected after 5 days of P4 vaginal treatment. Measures included serum P4 levels, ERA®-based evaluation of endometrial receptivity, and endometrial P4 levels along with metabolites (oestrone, oestradiol and 17α-hydroxyprogesterone) measured by ultra-performance liquid chromatography-tandem mass spectrometry. MAIN RESULTS AND THE ROLE OF CHANCE: Seventy-nine women were included (mean age: 39.9 ± 4.6, BMI: 24.2 ± 3.9 kg/m2, endometrial thickness: 8.2 ± 1.4 mm). The percentage of endometria indicated as receptive by ERA® was 40.5%. When comparing receptive versus non-receptive groups, no differences were observed in baseline characteristics nor in steroid hormones levels in serum or endometrium. No association between serum P4 and endometrial steroid levels or ERA result was found (P < 0.05). When the population was stratified according to metabolite concentration levels, endometrial P4 and 17α-hydroxyprogesterone were significantly associated with endometrial receptivity (P < 0.05). A higher proportion of receptive endometria by ERA was observed when endometrial P4 levels were higher than 40.07 µg/ml (relative maximum) and a lower proportion of receptive endometria was associated with endometrial 17α-hydroxyprogesterone lower than 0.35 ng/ml (first quartile). A positive correlation R2 = 0.67, P < 0.001 was observed between endometrial P4 and 17α-hydroxyprogesterone levels. LIMITATIONS, REASONS FOR CAUTION: This study did not analyse pregnancy outcomes. Further, the findings can only be extrapolated to HRT cycles with micronised vaginal progesterone for luteal phase support. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that the combined benefits of different routes of progesterone administration for luteal phase support could be leveraged to ensure an adequate concentration of progesterone both in the uterus and in the bloodstream. Further studies will confirm whether this method can optimise both endometrial receptivity and live birth rate. Additionally, targeted treatment to increase P4 endometrial levels may normalise the timing of the window of implantation without needing to modify the progesterone administration day. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the IVI-RMA Valencia (1706-VLC-051-EL) and Consellería d'Educació, Investigació, Cultura, i esport Generalitat Valenciana (Valencian Government, Spain, GV/2018//151). Almudena Devesa-Peiro (FPU/15/01398) and Cristina Rodriguez-Varela (FPU18/01657) were supported by the FPU program fellowship from the Ministry of Science, Innovation and Universities (Spanish Government). P.D.-G. is co-inventor on the ERA patent, with non-economic benefits. The other authors have no competing interests. TRIAL REGISTRATION NUMBER: NCT03456375.

Reproductive outcomes after hysteroscopic metroplasty for women with dysmorphic uterus and recurrent implantation failure.

BACKGROUND: The aim of this study was to assess the reproductive outcomes of women with recurrent implantation failure (RIF) after hysteroscopic metroplasty for dysmorphic uteri. METHODS: This retrospective observational study included 190 women with a diagnosis of RIF. These patients were eligible for hysteroscopic metroplasty for dysmorphic uteri, including T-shaped uteri, between January 2008 and September 2015 at the Instituto Valenciano de Infertilidad (IVI) in Valencia, Spain. RESULTS: The total clinical pregnancy rate, the live birth rate, and the abortion rate were 80.0% (152/190), 77.9% (147/190) and 8.9%, respectively. At 12 months, the clinical pregnancy rate was 76.3% (145/190) and at 6 months 50.5% (96/190). After the metroplasty, approximately 76% of all gravidities, were achieved during the first 12 months of follow-up. Within the first IVF cycle, pregnancy and live birth rates were 77.8% and 86.1%, respectively. The mean time to pregnancy was 6.5 months. CONCLUSION: This study demonstrates that hysteroscopic metroplasty improves pregnancy and live birth rates for women with a history of recurrent implantation failure and dysmorphic uterus. However, conclusions must be taken carefully as this is an observational study. A prospective, randomized and controlled study is necessary to support these results.

Low serum progesterone on the day of embryo transfer is associated with a diminished ongoing pregnancy rate in oocyte donation cycles after artificial endometrial preparation: a prospective study.

STUDY QUESTION: Is there a relationship between serum progesterone (P) and endometrial volume on the day of embryo transfer (ET) with ongoing pregnancy rate (OPR) in artificial endometrium preparation cycles? SUMMARY ANSWER: Patients with serum P < 9.2 ng/ml on the day of ET had a significantly lower OPR but endometrial volume was not related with OPR. WHAT IS KNOWN ALREADY: A window of optimal serum P levels during the embryo implantation period has been described in artificial endometrium preparation cycles. A very low endometrial volume is related to poor reproductive outcome. STUDY DESIGN, SIZE, DURATION: Prospective cohort study with 244 patients who underwent ET in an oocyte donation cycle after an artificial endometrial preparation cycle with estradiol valerate and vaginal micronized progesterone (400 mg/12 h). The study period went from 22 February 2016 to 25 October 2016 (8 months). Sample size was calculated to detect a 20% difference in OPR (35-55%) between two groups according to serum P levels in a two-sided test (80% statistical power, 95% confidence interval (CI)). PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients undergoing their first/second oocyte donation cycle, aged <50, BMI < 30 kg/m2, triple layer endometrium >6.5 mm and 1-2 good quality transferred blastocysts. A private infertility centre. Serum P determination and 3D ultrasound of uterine cavity were performed on the day of ET. Endometrial volume measurements were taken using a virtual organ computer-aided analysis (VOCAL™) system. The primary endpoint was OPR beyond pregnancy week 12. MAIN RESULTS AND ROLE OF CHANCE: About 211 of the 244 recruited patients fulfilled all the inclusion/exclusion criteria. Mean serum P on the day of embryo transfer was 12.7 ± 5.4 ng/ml (Centiles 25, 9.2; 50, 11.8; 75,15.8). OPRs according to serum P quartiles were: Q1: 32.7%; Q2: 49.1%; Q3: 58.5%; Q4: 50.9%. The OPR of Q1 was significantly lower than Q2-Q4: 32.7% versus 52.8%; P = 0.016; RR (95% CI): 0.62 (0.41-0.94). The mean endometrial volume was 3.4 ± 1.9 ml. Serum P on the day of ET did not correlate with endometrial volume. A logistic regression analysis, adjusted for all the potential confounders, showed that OPR significantly lowered between women with serum P < 9.2 ng/ml versus ≥9.2 ng/ml (OR: 0.297; 95%CI: 0.113-0.779); P = 0.013. The ROC curve showed a significant predictive value of serum P levels on the day of ET for OPR, with an AUC (95%CI) = 0.59 (0.51-0.67). LIMITATIONS, REASONS FOR CAUTION: Only the women with normal uterine cavity, appropriate endometrial thickness and good quality blastocysts transfer were included. Extrapolation to an unselected population or to other routes and/or doses of administering P needs to be validated. The role of endometrial volume could not be fully defined as very few patients presented a very low volume. WIDER IMPLICATIONS OF THE FINDINGS: The present study suggests a minimum threshold of serum P values on the day of ET that needs to be reached in artificial endometrial preparation cycles to optimize outcome. No upper threshold could be defined. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: NCT02696694.

The follicular hormonal profile in low-responder patients undergoing unstimulated cycles: Is it hypoandrogenic?

STUDY QUESTION: What is the final hormonal milieu of pre-ovulatory follicles of low-responder (LR) patients undergoing unstimulated cycles? SUMMARY ANSWER: Neither androgen secretion nor LH was impaired in pre-ovulatory follicles of LR women. WHAT IS KNOWN ALREADY: Therapies currently used to improve ovarian response in LR women have an impact on the final hormonal follicular milieu, and these changes are believed to be partially responsible for determining the success rate in these women. Surprisingly, as far as we know, there is no report of the final hormonal profile of LR women undergoing unstimulated cycles or evidence that follicular androgen secretion in LR women is impaired. STUDY DESIGN, SIZE AND DURATION: A prospective case-control study including 94 women, 36 normal controls and 58 LR patients (19 Young ≤ 35 years LR and 39 Aged >35 years LR) from 2009 to 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Fifty-eight LR women were divided into two groups: Young LR (age ≤ 35; n = 19) and Aged LR (ALR; age >35; n = 39). The control group (group C) comprised 36 egg donors undergoing an unstimulated cycle in our IVF unit. Serum and follicular fluid hormonal concentrations for estradiol (E2), progesterone, testosterone and androstendione were measured. The spindle parameters of metaphase II oocytes generated from these groups were also analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Pre-ovulatory follicles from LR patients had similar androgenic and LH concentrations to those observed in the control group. However, higher intrafollicular concentrations of FSH and progesterone were observed in ALR. Moreover, no differences were found for the spindle evaluation of oocytes between groups by the Oosight technology. LIMITATIONS, REASONS FOR CAUTION: The controls were younger and had a lower BMI than the LR women. The sample size available restricted statistical power. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that the problem with LR women is not the final pre-ovulatory follicular androgen concentration since this is similar to normal responders, but in the ability to respond to controlled ovarian stimulation protocols. Therefore, efforts should be focused on long-interval androgen priming to potentially increase the recruitment of small antral follicles rather than increasing the intraovarian androgen levels within the current cycle. STUDY FUNDING/COMPETING INTEREST: The present project has been supported by the R+D programme from the Generalitat Valenciana (Regional Valencian Government) IMPIVA MIDTF/2010/95. The authors have no conflict of interest to declare.

Endometrial receptivity is affected in women with high circulating progesterone levels at the end of the follicular phase: a functional genomics analysis.

BACKGROUND: Elevated serum progesterone levels at the end of the follicular phase in controlled ovarian stimulation (COS) leads to a poorer ongoing pregnancy rate in IVF cycles due to reduced endometrial receptivity. The objective of this study was to use microarray technology to compare endometrial gene expression profiles at the window of implantation according to the levels of circulating progesterone. METHODS: For this prospective cohort study, microarray data were obtained from endometrial biopsies from 12 young healthy oocyte donors undergoing COS with pituitary suppression by either gonadotrophin-releasing hormone (GnRH) agonists or antagonists, and recombinant FSH. On the day of recombinant chorionic gonadotrophin (rCG) administration, six women had serum progesterone levels (P) >1.5 ng/ml (study group) and six had serum P levels <1.5 ng/ml (control group). Endometrial samples were collected using a Pipelle catheter 7 days after the rCG injection. RESULTS: Using the parametric test, we identified 140 genes significantly dysregulated (64 up- and 76 down-regulated) in the study group endometria compared with the control endometria, regardless of the GnRH analogue employed. These genes are related to cell adhesion, developmental processes, the immune system and others, which are all required for normal endometrial function development. Of the 25 gene targets previously proposed as markers for endometrial receptivity, 13 appeared over-regulated in the study group. CONCLUSIONS: Our results reveal that elevated progesterone levels on the day of rCG administration can induce significant alterations in the gene expression profile of the endometrium.

Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: analysis of over 4000 cycles.

BACKGROUND: The influence of elevated serum progesterone levels during in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles on pregnancy rates is a matter of continued debate among fertility clinicians. Efforts to resolve this question have been impeded by the various assays used to measure progesterone and the different, arbitrary threshold values for defining 'high' progesterone levels. METHODS: A non-interventional, retrospective, observational, single-centre cohort study evaluated the relationship between serum progesterone levels on the day of human chorionic gonadotrophin (hCG) administration and the ongoing pregnancy rate in 4032 patients undergoing IVF/ICSI cycles using gonadotrophin-releasing hormone (GnRH) analogues for pituitary down-regulation. RESULTS: Ongoing pregnancy rates were inversely associated with serum progesterone levels on the day of hCG (P < 0.001 for overall trend), irrespective of the GnRH analogue used for pituitary down-regulation. Patients with serum progesterone levels < or = 1.5 ng/ml had significantly higher ongoing pregnancy rates than those with progesterone levels >1.5 ng/ml (31.0 versus 19.1%; P = 0.00006); odds ratio, 0.53 (95% confidence interval, 0.38-0.72). Multivariate regression analysis showed that daily follicle-stimulating hormone dose, number of oocytes and estradiol values on the day of hCG administration were positively associated with progesterone levels (P < 0.0001 for all). Serum progesterone levels were significantly greater in women treated with GnRH agonists (n = 1177) versus antagonists (n = 2855; 0.84 +/- 0.67 versus 0.75 +/- 0.66 ng/ml; P = 0.0003). CONCLUSIONS: Elevated serum progesterone levels on the day of hCG administration is associated with reduced ongoing pregnancy rates. In particular, serum progesterone levels of >1.5 ng/ml were associated with lower ongoing pregnancy rates following IVF/ICSI cycles irrespective of the GnRH analogue used for pituitary down-regulation.

Highly purified hMG versus recombinant FSH in ovarian hyperstimulation with GnRH antagonists--a randomized study.

BACKGROUND: Highly purified hMG (hp-hMG) has recently shown better cycle outcome than the recombinant FSH (rFSH) when compared in GnRH agonist long protocol cycles. However, they have not yet been compared in GnRH antagonist cycles. METHODS: A RCT comparing the ongoing pregnancy rate (primary end-point) in 280 patients undergoing IVF/ICSI after stimulation with hp-hMG or rFSH controlled with a GnRH antagonist. RESULTS: No significant differences were observed between hp-hMG and rFSH in terms of the ongoing pregnancy rate per started cycle (35.0 versus 32.1%, respectively; P = 0.61); relative risk: 1.09 (95% confidence interval: 0.78-1.51; risk difference: 2.9%). No differences were observed for implantation, clinical pregnancy and pregnancy loss rates. More oocytes were obtained from patients receiving rFSH then hMG (14.4 +/- 8.1 versus 11.3 +/- 6.0, respectively; P = 0.001). Estradiol was higher at the end of stimulation in the hp-hMG group (P = 0.02), whereas progesterone was higher in patients stimulated with rFSH (P < 0.001). CONCLUSIONS: A similar outcome was observed for hp-hMG and rFSH when used for stimulation in GnRH antagonist cycles. However, some differences were found in ovarian response in terms of oocyte yield and hormonal profile. Clinical Trials.gov TRIAL REGISTRATION NUMBER: NCT00669786.

Rotura uterina y cesárea anterior. Revisión y casuística durante el período de 1999 a 2002 en el Hospital Universitario La Paz / Uterine rupture and prior cesarean section. Review of casuistics (1999-2002) in La Paz University Hospital

Objetivos: Revisión de las roturas uterinas (RU) y su relación con la existencia de una cesárea previa, ocurridas en los últimos años en el Hospital La Paz. Material y métodos: Se han revisado las historias clínicas de las pacientes que han tenido una rotura uterina intraparto entre los años 1999-2002. Se ha recogido el número de pacientes con cesárea anterior (CA) y la vía de finalización del parto, y se ha calculado la incidencia de RU total y en mujeres con cesárea anterior. Resultados: Durante el período de estudio se produjeron 18 RU sobre un total de 35.323 (0,05 por ciento) partos. Se atendieron 2.207 partos en mujeres con cesárea anterior, con una incidencia de RU de 0,49 por ciento. Las mujeres con CA a las que se realizó una cesárea electiva tuvieron una incidencia de RU de 0,44 por ciento. En las que se intentó el parto por vía vaginal la frecuencia de RU fue de 0,53 por ciento. Conclusión: La RU es más frecuente en mujeres con CA que sin ella. El mayor riesgo corresponde a aquellas con 2 o más cesáreas previas, y es similar entre aquellas con cesárea electiva o intento de parto por vía vaginal, siempre que se respeten las indicaciones generales para intentar el parto vaginal (AU)

Masa ósea en pediatría. Valoración por métodos incruentos: radiogrametría metacarpiana / Bone mass in pediatrics. Assessment by non-invasive methods: metacarpal radiogrammetry

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