The effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles.

PURPOSE: Does vitrification/warming affect the mitochondrial DNA (mtDNA) content and the gene expression profile of blastocysts? METHODS: Prospective cohort study in which 89 blastocysts were obtained from 50 patients between July 2017 and August 2018. mtDNA was measured in a total of 71 aneuploid blastocysts by means of real-time polymerase chain reaction (RT-PCR). Transcriptomic analysis was performed by RNA sequencing (RNA-seq) in an additional 8 aneuploid blastocysts cultured for 0 h after warming, and 10 aneuploid blastocysts cultured for 4-5 h after warming. RESULTS: A significant decrease in mtDNA content just during the first hour after the warming process in blastocysts was found (P < 0.05). However, mtDNA content experimented a significantly increased along the later culture hours achieving the original mtDNA levels before vitrification after 4-5 h of culture (P < 0.05). Gene expression analysis and functional enrichment analysis revealed that such recovery was accompanied by upregulation of pathways associated with embryo developmental capacity and uterine embryo development. Interestingly, the significant increase in mtDNA content observed in blastocysts just after warming also coincided with the differential expression of several cellular stress response-related pathways, such as apoptosis, DNA damage, humoral immune responses, and cancer. CONCLUSION: To our knowledge, this is the first study demonstrating in humans, a modulation in blastocysts mtDNA content in response to vitrification and warming. These results will be useful in understanding which pathways and mechanisms may be activated in human blastocysts following vitrification and warming before a transfer.

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.

Obesity Affects Endometrial Receptivity by Displacing the Window of Implantation.

Our aim was to determine prospectively whether increased body mass index (BMI) affects endometrial receptivity through displacement of the window of implantation (dWOI) using the endometrial receptivity analysis (ERA), and whether this effect is BMI-dependent. We recruited a population of 170 infertile women with a normal uterus and no clinical history of recurrent miscarriage or implantation failure. These women were divided into four groups according to BMI: normal weight (18.5-24.9 kg/m2; n = 44), overweight (25-29.9 kg/m2; n = 29), class I obese (30.0-34.9 kg/m2; n = 54), and class II and III obese (> 35 kg/m2; n = 43). We also assigned the patients to one of two larger BMI cohorts: non-obese (normal weight and overweight; n = 73) and obese (class I, II, and III obese; n = 97). We compared analytical and clinical data and dWOI in these categories, finding significant metabolic differences in glycemia, TSH, insulin, HDL cholesterol, LDL cholesterol, triglycerides, and systolic and diastolic blood pressure among the different BMI groups. One-day dWOI increased significantly with BMI, and significant differences were observed in the non-obese versus obese categories (9.7% vs 25.3 %, respectively (p = 0.02)). dWOI was most pronounced in patients with class II-III obesity. In addition, displacement was longer as BMI increased since ERA revealed a higher proportion of displacements of 1 day than of 12 h and showed they were predominantly pre-receptive. In conclusion, obesity has a negative effect on endometrial receptivity through delaying of the WOI, which increases in function of BMI as well as the metabolic disturbances of the patient.

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.

A 5-year multicentre randomized controlled trial comparing personalized, frozen and fresh blastocyst transfer in IVF.

RESEARCH QUESTION: Does clinical performance of personalized embryo transfer (PET) guided by endometrial receptivity analysis (ERA) differ from frozen embryo transfer (FET) or fresh embryo transfer in infertile patients undergoing IVF? DESIGN: Multicentre, open-label randomized controlled trial; 458 patients aged 37 years or younger undergoing IVF with blastocyst transfer at first appointment were randomized to PET guided by ERA, FET or fresh embryo transfer in 16 reproductive clinics. RESULTS: Clinical outcomes by intention-to-treat analysis were comparable, but cumulative pregnancy rate was significantly higher in the PET (93.6%) compared with FET (79.7%) (P = 0.0005) and fresh embryo transfer groups (80.7%) (P = 0.0013). Analysis per protocol demonstrates that live birth rates at first embryo transfer were 56.2% in PET versus 42.4% in FET (P = 0.09), and 45.7% in fresh embryo transfer groups (P = 0.17). Cumulative live birth rates after 12 months were 71.2% in PET versus 55.4% in FET (P = 0.04), and 48.9% in fresh embryo transfer (P = 0.003). Pregnancy rates at the first embryo transfer in PET, FET and fresh embryo transfer arms were 72.5% versus 54.3% (P = 0.01) and 58.5% (P = 0.05), respectively. Implantation rates at first embryo transfer were 57.3% versus 43.2% (P = 0.03), and 38.6% (P = 0.004), respectively. Obstetrical outcomes, type of delivery and neonatal outcomes were similar in all groups. CONCLUSIONS: Despite 50% of patients dropping out compared with 30% initially planned, per protocol analysis demonstrates statistically significant improvement in pregnancy, implantation and cumulative live birth rates in PET compared with FET and fresh embryo transfer arms, indicating the potential utility of PET guided by the ERA test at the first appointment.

Mitochondrial DNA content decreases during in vitro human embryo development: insights into mitochondrial DNA variation in preimplantation embryos donated for research.

OBJECTIVE: To assess the mitochondrial DNA (mtDNA) load and variation in human oocytes and during preimplantation embryo development using specimens donated for research. DESIGN: Prospective cohort study. SETTING: Not applicable. PATIENTS: A total of 50 in vitro fertilization patients and 11 oocyte donors whose specimens were obtained between July 2017 and July 2018. INTERVENTIONS: None. MAIN OUTCOME MEASURES: All specimens were separately collected. Quantitative polymerase chain reaction was performed with SurePlex DNA Amplification System (Illumina). Primers for the adenosine triphosphate 8 mitochondrial gene and the ß-actin were used. Data were statistically analyzed by analysis of variance with the Scheffé multiple pairwise comparison for categorical variables and by linear regression for numerical variables. RESULTS: Human metaphase II (MII) oocytes had significantly more total mtDNA copy number than day 3 embryos, and day 3 embryos had more total and per-cell mtDNA copy number than aneuploid blastocysts. There was a significant decrease in mtDNA content associated with failed-fertilized oocytes compared to noninseminated metaphase II oocytes. CONCLUSIONS: During preimplantation development, before implantation, human embryos undergo a significant decrease in total mtDNA content and no increase in mtDNA content at the blastocyst stage. Oocytes need to carry a correct threshold of mitochondrial load in the oocyte in order to successfully fertilize. An active degradation of mtDNA before implantation occurs after fertilization takes place. These findings could be used to improve knowledge about the best embryo culture conditions and would serve as a basis for further studies addressing again the use of mtDNA content as an embryo viability marker.

In vitro fertilization with preimplantation genetic diagnosis for aneuploidies in advanced maternal age: a randomized, controlled study.

OBJECTIVE: To determine the clinical value of preimplantation genetic diagnosis for aneuploidy screening (PGD-A) in women of advanced maternal age (AMA; between 38 and 41 years). DESIGN: This was a multicenter, randomized trial with two arms: a PGD-A group with blastocyst transfer, and a control group with blastocyst transfer without PGD-A. SETTING: Private reproductive centers. PATIENT(S): A total of 326 recruited patients fit the inclusion criteria, and 205 completed the study (100 in the PGD-A group and 105 in the control group). INTERVENTION(S): Day-3 embryo biopsy, array comparative genomic hybridization, blastocyst transfer, and vitrification. MAIN OUTCOME MEASURE(S): Primary outcomes were delivery and live birth rates in the first transfer and cumulative outcome rates. RESULT(S): The PGD-A group exhibited significantly fewer ETs (68.0% vs. 90.5% for control) and lower miscarriage rates (2.7% vs. 39.0% for control). Delivery rate after the first transfer attempt was significantly higher in the PGD-A group per transfer (52.9% vs 24.2%) and per patient (36.0% vs. 21.9%). No significant differences were observed in the cumulative delivery rates per patient 6 months after closing the study. However, the mean number of ETs needed per live birth was lower in the PGD-A group compared with the control group (1.8 vs. 3.7), as was the time to pregnancy (7.7 vs. 14.9 weeks). CONCLUSION(S): Preimplantation genetic diagnosis for aneuploidy screening is superior compared with controls not only in clinical outcome at the first ET but also in dramatically decreasing miscarriage rates and shortening the time to pregnancy.

A time to look back: analysis of morphokinetic characteristics of human embryo development.

OBJECTIVE: To describe the times associated with the morphological changes that occur in the embryo during preimplantation development based on the largest sample size described with time lapse. DESIGN: Cohort study. SETTING: University-affiliated private center. PATIENT(S): A total of 9,530 embryos from 1,806 intracytoplasmic sperm injection (ICSI) cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Using a time-lapse system, embryo images were acquired for at least 68 hours, in some cases reaching 120-130 hours. Embryo cleavage time points up to 8-cell-stage (t2-t8) as well as morulae (tM) and blastocyst formation (tB) were registered in hours after ICSI. Additionally, duration of the cell cycle (cc) and synchrony (s) of the second and third cell cycles were defined. Finally, four subgroups of embryos were considered: the "regular divisions" group excluded embryos with a direct cleavage from 1 to 3 or 2 to 5 cells, and the "viable 8-cell," the "viable blastocyst," and "implanted embryos" groups included only embryos viable to the 8-cell stage, blastocyst stage, or transferred and successfully implanted, respectively. RESULT(S): Averages of times in the general population were: t2 = 27.9 hours, t3 = 38.2 hours, t4 = 40.7 hours, t5 = 51.0 hours, t6 = 54.1 hours, t7 = 56.7 hours, t8 = 59.1 hours, tM = 86.6 hours, tB = 104.1 hours, cc2 = 10.3 hours, cc3 = 12.8 hours, s2 = 2.7 hours, and s3 = 9.9 hours. Comparison between groups showed significant differences between regular divisions and viable 8 cells for t2, t3, t5, cc2, cc3, s2, and s3; between 8 cells and blastocyst for t5, t8, tM, cc3, and s2; and between blastocyst and implanted embryos for t8, tM, tB, and s2. Differences in timing related to morphology of cleavage- and blastocyst-stage embryos were detected. CONCLUSION(S): A time-lapse monitoring system applied to embryology allows accuracy and objectivity when defining the basis of embryo development within a clinic. The sample size is the largest ever described that provides consistent information about the normal distribution of embryo developmental timings.

GnRH agonist administration at the time of implantation does not improve pregnancy outcome in intrauterine insemination cycles: a randomized controlled trial.

OBJECTIVE: To assess whether GnRH agonist administration in the luteal phase improves pregnancy outcome in intrauterine insemination (IUI) cycles. DESIGN: Single-center, randomized, single-blind, placebo-controlled trial. SETTING: University-affiliated infertility clinic, between February 2005 and December 2007. PATIENT(S): Three hundred forty-four women undergoing IUI owing to mild to moderate male factor or donor sperm indication. INTERVENTION(S): Random administration to either a single subcutaneous injection of 0.1 mg triptorelin (group A; n=172) 8 days after hCG administration, or solvent only (group B; n=172) at the same time. MAIN OUTCOME MEASURE(S): Pregnancy rate was the primary outcome measure considered for assessing the role of triptorelin administration at the time of implantation. Clinical pregnancy, miscarriage, and ongoing pregnancy rates were the secondary outcome measures. RESULT(S): No differences were detected between the groups regarding clinical, seminal, or ovarian stimulation parameters. Pregnancy rate per randomized patient was similar in both groups (22.7% vs. 22.1%), as were clinical pregnancy, miscarriage, and ongoing pregnancy rates. There was a significant increase in the proportion of multiple pregnancies in the placebo group (10.3% vs. 36.8%). CONCLUSION(S): Administration of GnRH agonist at the time of implantation does not improve the reproductive outcome of IUI cycles.