Analysis Of Endometrial Biopsies Research Articles

P–261 The human embryo following biopsy on day5 vs day3: Implantation, cytoskeleton, ultrastructure and effects of endometrial damage/inflammation on receptivity as revealed by scanning electron microscopy

Abstract Study question Are there any differences in implantation, cytoskeleton and ultrastructure of embryos biopsied on day5 vs day3 and how endometrial damage/inflammation may affect receptivity and implantation? Summary answer No differences are observed in implantation rates but vitrification following day5 biopsy led to more cytoskeletal/ultrastructural anomalies. Infections and epithelial damage severely affected endometrial receptivity. What is known already Successful implantation is dependent on the correct synchronization of the window of implantantation with the transfer of chromosomally/genetically normal embryos, in a well prepared receptive endometrium. This is the first study to examine the effects of day5 vs day3 embryo biopsy by comparing implantation/pregnancy rates and by analysing cytoskeleton using Confocal Laser Scanning Microscopy (CLSM), and ultrastructure by Transmission Electron Microscopy (TEM). In addition, Scanning Electron Microscopy (SEM) was used on endometrial biopsies to assess possible uterine pathologies/inflammation that may be responsible for the failed implantation after PGT-A/M and if subsequent treatment can increase implantation/pregnancy rates in succeeding PGT-A/M cycles. Study design, size, duration 470 embryos were biopsied on day5 for PGT-A (n = 152–37 cycles) or on day3 for PGT-A (n = 162–29 cycles) and PGT-M (n = 156–22 cycles). Following transfer of normal embryos, spare embryos, rejected for transfer following day5 or day3 biopsy were processed for Cytoskeletal analysis (n = 30 fresh day3 biopsied, n = 30 day5 biopsied/vitrified) or TEM (n = 20 fresh day3 biopsied, n = 20 day5 biopsied/vitrified). Also, patients with a –ve hCG test, underwent endometrial biopsy to detect infection/inflammation and assess receptivity. Participants/materials, setting, methods Cytoskeletal analysis was performed by embryo immunostaining with α-tubulin, γ-tubulin, acetylated-tubulin antibodies and DAPI or/ PI to visualise DNA. TEM analysis was carried out following embryo fixation in gluteraldehyde, incubation in osmium, aqueous uranyl acetate, dehydration through ethanol series, and immersion in Epon. Endometrial biopsies were fixed in gluteraldehyde solution and processed for SEM using standard methods. The study was conducted in an academic hospital with an IVF/PGD laboratory and 3 private IVF Units. Main results and the role of chance 162 embryos were biopsied on day 3 for PGT-A (29 cycles), 10 cycles had no normal embryos for transfer, 30 normal embryos were transferred in 19 cycles leading to 12/19 (63.2%) +vehCG/ET and 11/19 (57.9%) Ongoing pregnancy rate/ET. 156 embryos were biopsied on day 3 for PGT-M (22 cycles), 2 cycles had no normal embryos for transfer, 34 normal or carrier embryos were transferred in 20 cycles leading to 15/20 (75.0%) +vehCG/ET and 13/20 Ongoing pregnancy rate/ET (65.0%). 152 embryos were biopsied on day 5 for PGT-A (37 cycles), 8 cycles had no normal embryos for transfer, 34 normal embryos were transferred in 29 cycles leading to 18/29 (62.1%) +vehCG/ET and 16/29 (55.2%) Ongoing pregnancy rate /ET. Analysis of endometrial biopsies with SEM revealed bacterial infections, inflammation and epithelial damage. So far, 33.3% of patients who received intracavitary infusions-antibiotic treatment per os achieved a +vehCG/ET in their next PGT-A/M cycle. Cytoskeletal analysis showed that the majority of spindles examined in both day3 and day5 biopsied embryos were normal (85/114(74.6%) and 87/137(63.5). However vitrification following day5 biopsy led to more cytoskeletal/ultrastructural anomalies which included multipolar/abnormally shaped spindles, chromosome-bridging, chromosome-lagging and more vacuoles, lipofuscins. and distension of mitochondria. Limitations, reasons for caution Patients undergoing PGT-A have various aetiologies (heterogeneous group). The embryos used for cytoskeletal and ultrastructural analysis in this study were all diagnosed with either chromosomal abnormalities or single gene defects following PGT-A or PGT-M. Wider implications of the findings: This is the first study to compare implantation/pregnancy rates, cytoskeleton and ultrastructure of day5 vs day3 biopsied embryos. The similarities observed in implantation/pregnancy rates, and the limited ultrastuctural and cytoskeletal anomalies identified confirm the procedures’ safety and indicate in certain cases endometrial factors/inflammation responsible for failed implantation following PGT-A/M. Trial registration number Not applicable

Recommendation for the collection and analysis of endometrial biopsies for hormone therapies

ABSTRACTThe assessment of endometrial safety is one of the key requirements for the clinical development of new products for hormone therapy (HT) to treat menopausal symptoms in women who have a uterus. Both the Food and Drug Administration (FDA) in the United States and the European Medicines Agency (EMA) provide detailed guidance on the requirements for the evaluation of biopsies to prove endometrial safety. However, there are some discrepancies between the European and the US requirements, making it difficult to fulfil both guidelines simultaneously.In order to facilitate multinational clinical trials performed within clinical programs to develop novel HT products, we developed an approach considering both guidance documents as far as possible and proposed solutions for issues that are inconsistently described in these guidelines. A table with the required sample sizes is given.Our recommendation for a unified approach for the estimation of the hyperplasia rate for hormone therapies fulfils the intent of the recommendations of both the FDA and the EMA and thus leads to a globally harmonized drug development for hormone therapies.

Messenger ribonucleic acid encoding interferon-inducible guanylate binding protein 1 is induced in human endometrium within the putative window of implantation.

The putative window of embryo implantation in the human opens between days 19--24 of the menstrual cycle. During this period, the endometrium undergoes distinctive structural and functional changes orchestrated by steroid hormones, growth factors, and cytokines to attain a receptive phase in which it acquires the ability to implant the developing embryo. A major challenge in the study of human reproduction is to identify the molecular signals that participate in the establishment of this critical receptive phase in the context of the natural cycle. Toward this goal, we analyzed human endometrial biopsies at various days of the menstrual cycle by employing messenger RNA (mRNA) differential display technique. We isolated several complementary DNAs representing genes that are either up- or down-regulated within the putative window of implantation. We identified one of these genes as that encoding interferon (IFN)-inducible guanylate-binding protein 1 (or GBP1), which possesses GTPase activity. Analysis of endometrial biopsies by Northern blotting and RT-PCR demonstrated that GBP1 mRNA is specifically induced at the midsecretory phase of the menstrual cycle. In situ hybridization analysis revealed that GBP1 mRNA expression is localized in the glandular epithelial cells as well as in the stroma in the immediate vicinity of the glands. We observed that treatment of human endometrial adenocarcinoma cell, Ishikawa, with IFN-gamma or IFN-alpha markedly induced the expression of GBP1 mRNA. IFN-gamma was, however, a more potent inducer of GBP1 than IFN-alpha. Consistent with this finding, the temporal profile of GBP1 expression during the menstrual cycle resembled that of IFN-gamma mRNA more closely than that of IFN-alpha, predicting a regulatory role of IFN-gamma in GBP1 expression in midsecretory human endometrium. Although the precise function of GBP1 in the receptive human uterus remains unclear, its unique expression overlapping the putative window of implantation suggests that it might serve as a useful marker of uterine receptivity in the human.