Transfers, Embryo

Embryo collection was performed in a surgical room located on the farm. The donors were subjected to a midventral laparotomy on Days 5 and 6 of the estrous cycle (Day 0: onset of estrus) to obtain morulae and unhatched blastocysts, respectively. The donors were sedated by the administration of azaperone (2 mg/kg body weight, intramuscular). General anesthesia was induced using sodium thiopental (7 mg/kg body weight, intravenous) and maintained with isoflurane (3.5–5%). After exposure of the genital tract, the corpora lutea on the ovaries were counted. Embryos were collected by flushing the tip of each uterine horn with 30 mL of a chemically defined medium consisting of Tyrode's lactate (TL)-HEPES-polyvinyl alcohol (PVA) [17] (link) with some modifications. This medium (TL-PVA) was composed of 124.3 mM NaCl, 3.2 mM KCl, 2 mM NaHCO₃, 0.34 mM KH₂PO₄, 10 mM Na-lactate, 0.5 mM MgCl₂·6H₂O, 2 mM CaCl₂·2H₂O, 10 mM HEPES, 0.2 mM Na-pyruvate, 12 mM sorbitol, 0.1% (w/v) PVA, 75 µg/ml potassium penicillin G and 50 µg/mL streptomycin sulfate. The collected embryos were evaluated to verify their developmental stage and quality grade. One-cell eggs and poorly developed embryos were classified as oocytes and degenerate embryos, respectively. The remaining embryos that exhibited appropriate morphology according to the criteria determined by the International Embryo Transfer Society [18] were considered viable. Only compacted morulae and unhatched blastocysts graded as excellent or good based on morphological appearance were used in the experiments according to the specific experimental design.
The ovulatory response of the donors was determined by counting the number of corpora lutea in both ovaries. To evaluate the effectiveness of the superovulation treatment, the numbers of viable embryos and oocytes and degenerate embryos were counted in each donor. The recovery rate was defined as the ratio of the number of embryos and oocytes and degenerate embryos recovered to the number of corpora lutea present. The fertilization rate was defined as the ratio of the number of viable embryos to the total number of embryos and oocytes and degenerate embryos collected.

All animal procedures were carried out in accordance with UK legal requirements and in under licensed approval from the UK Home Office. In the current study a mouse model of menstruation described by Brasted et al [15] (link) was modified to include non-surgical induction of decidualisation and a longer decidualisation period. Uterine tissues were also collected during a period of active shedding and repair, time-points that have not been previously described.
On day 0, C57BL/6J mice between 8–10 weeks of age were ovariectomised to deplete endogenous steroid production. Mice received daily injections of β-oestradiol (E2) in sesame seed oil (100 ng/100 µl, days 7–9). A progesterone (P4)-secreting pellet was placed sub-cutaneously on day 13; mice also received daily injections of sub-cutaneous injections of E2 (5 ng/100 µl, days 13–15). On day 15, decidualisation of one uterine horn was induced by stimulation of the horn using sesame seed oil (20 µl) inserted into the uterine lumen via the cervix using a non-surgical embryo transfer device (NSET) from Datesand Ltd. (Manchester, UK). The contra-lateral horn acted as a control. P4 withdrawal was induced 90 hours after decidualisation by removing the P4-pellet. Mice were culled by asphyxiation and cervical dislocation at time of P4 withdrawal or 4, 8, 12, 24 and 48 hours thereafter (Figure 1). Mice received an intra-peritoneal injection of bromodeoxyuridine (BrdU, 2.5 mg/ml) 90 minutes prior to culling to detect cellular proliferation. Blood sera were collected, uteri dissected and collected into RNA later or 4% neutral buffered formalin. Any mouse in which the oil-treated horn had not decidualised was excluded from the study.

We input “recurrent implantation failure” and “expression profile” as two keywords to the GEO database, then two datasets GSE103465 and GSE111974 were selected for analysis, and GSE26787 was chosen for validation. Both GSE103465 and GSE111974 contain expression profiles of endometrial tissue obtained from RIF and control women during WOI. GSE103465, in GPL16043 platform, contains whole-genome expression profiles of endometrial tissue from three women divided to the control group and RIF group (Guo et al., 2018 (link)). In GSE103465, RIF is defined as no pregnancy after ≥3 embryo transfers including a total of ≥4 good-quality embryos, and inclusion criteria of control group is infertile women with tubal factors who achieved a clinical pregnancy after the first embryo transfer (Guo et al., 2018 (link)). GSE111974, in GPL17077 platform, consists of 24 individuals with RIF and 24 fertile control patients, in which RIF is determined as failure of pregnancy in three consecutive IVF cycles with at least one transfer of good quality embryo in each cycle, while the fertile control refers to patients who had a history of at least one live birth with no related comorbidities (Bastu et al., 2019 (link)). The platform and series matrix files were all downloaded.