{"paper_id":"4d469d8d-1da1-4a8e-a89a-01a21754f785","body_text":"Hyaluronan-enriched transfer medium (HETM) can improve the implantation rate in morphologically poor euploid blastocyst transfer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hyaluronan-enriched transfer medium (HETM) can improve the implantation rate in morphologically poor euploid blastocyst transfer KOJI NAKAGAWA, Takashi Horikawa, Yuji Orita, Emi Yamashiro, Hideaki Watanabe, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2427879/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 May, 2023 Read the published version in Archives of Gynecology and Obstetrics → Version 1 posted 5 You are reading this latest preprint version Abstract Purpose: Hyaluronan-enriched transfer medium (HETM) could improve the clinical pregnancy rate (CPR) for patients with repeated implantation failures (RIF). In contrast, there have been seldom reports addressing the potentially beneficial effects of HETM for morphologically poor blastocysts (MPBLs). Our study aimed to evaluate whether the use of HETM would improve the CPR for the patients who were transferred with euploid MPBLs. Methods: Patients who underwent single euploid blastocyst transfer between July 2020 and June 2022 were enrolled. We included only those blastocysts confirmed as euploid by PGT-A, and those blastocysts were transferred after thawing. The natural ovulatory cycle or hormone replacement cycle (HRC) protocol were used for endometrial preparation for thawed blastocyst transfer (thawed-BT). A total of 1,168 thawed-BT cycles were performed in the study period, including 954 cycles of morphologically good blastocysts (≥ 4BB in Gardner’s classification), and 85 cycles of MPBLs, of which 47 were transferred using HETM in thawed-BT (the HETM group), and the remaining 38 were transferred with the medium without hyaluronan (the control group). We compared the CPR between these two groups. Results: The characteristics of patients were similar between the HETM and control groups. The CPR in the HETM group was significantly higher than the control group (47.4% and 21.5%, respectively, p = 0.019). The multiple logistic regression analysis found that the use of HETM was a predictive factor of positive pregnancy outcomes (OR = 5.08, 95% CI = 1.62-16.0, p = 0.019). Conclusion: Our data suggests that HETM used in the euploid blastocyst transfer can improve the clinical pregnancy rates of morphologically poor blastocysts. Euploid blastocyst Hyaluronan HETM morphologically poor blastocyst pregnancy rate Figures Figure 1 Figure 2 Introduction Modern assisted reproductive technology (ART) treatments consist of various processes, starting from ovarian stimulation, oocyte pick-up, insemination, embryo culture and finally embryo transfer. Hyaluronan-enriched transfer medium (HETM) has been clinically used in many ART clinics as a medium specially formulated for embryo transfer, which was first reported in 2002 to improve the implantation rates of human embryos in ART treatment [ 1 ], followed by many other RCT studies demonstrating the efficacy of HETM in human embryo transfer [ 2 – 4 ]. Reported by Urman et al., HETM was effective in improving the implantation rates in a fresh embryo transfer cycle for those patients with history of one or more implantation failures and patients of advanced maternal ages [ 4 ]. Our group has also previously reported that the clinical pregnancy rates of those patients with repeated implantation failures (RIF) was improved in the thawed embryo transfer cycles using HETM [ 5 ]. Having this experience, our group have used HETM as the transfer medium for those patients with applicable histories or indications, or those who made a specific request after having an adequate consultation with the clinicians. More recently, a Cochrane review published in 2020, which cited data reported from a number of previous clinical studies using HETM in embryo transfer, supported the addition of hyaluronic acid (HA) in embryo transfer medium to improve the clinical outcomes of ART treatments, including the improvements in live births per ART trteatments and the decrease the rates of miscarriage [ 6 ]. Interestingly, we noted that many previous studies had selected embryos with good morphological grades for transfer, because in general poor morphological grades were thought to indicate lower implantation potential. More recently, preimplantation genetic testing for aneuploidy (PGT-A) is becoming commonplace, and it has been widely implemented in the treatment for patients with RIF or recurrent pregnancy losses, to reduce the chance of another failure in embryo transfer. While blastomere biopsy used to be a common procedure for PGT-A, nowadays it has been replaced almost entirely with trophectoderm (TE) biopsy taken at blastocyst stage [ 7 ]. In Japan, the selection of euploid blastocysts for transfer following PGT-A has been allowed since 2019. Recently, we reported that the morphologically good blastocysts had higher clinical pregnancy rates than morphologically poor blastocysts even when we only selected euploid blastocysts for transfer [ 8 ]. But there have been seldom clinical reports specially asking how HETM could improve the clinical pregnancy rates of blastocysts with poor morphological grades, or enriched hyaluronan could potentially help the implantation of a morphologically poor blastocyst. Therefore, we designed our study to determine whether the use of HETM improves the clinical pregnancy rate (CPR) of patients who underwent euploid blastocyst transfer with poor morphological grade. Selecting only euploid blastocysts by PGT-A, our study specially addresses the relationship between the embryo morphology and the transfer success rates in the presence of high concentration HA in embryo transfer medium. Materials And Methods Study design We conducted a single center, cross-sectional study between July 2020 and June 2022, which recruited a total of 1,168 thawed blastocyst transfers (thawed BT) at Sugiyama Clinic Shinjuku, Tokyo, Japan. A flowchart of the patient selection process is shown in Fig. 1 . All blastocysts used in this study were called as euploid or mosaic based on PGT-A results. Those blastocysts showing 20% or less of mosaicism or segmental mosaicism were called as euploid and accepted to thawed BT [ 9 ]. One hundred twenty-nine thawed BT were performed with mosaic blastocysts, while the remaining 1,039 thawed BTs were performed with euploid blastocysts. All transferred embryos had been subjected to morphological assessments after thawing, which resulted in 954 blastocysts with good morphological grades and 85 blastocysts with poor morphological grades available to thawed BTs. Of the 85 morphologically poor euploid blastocysts, 47 of these were used for HETM and the remaining 38 were transferred without HETM at thawed BT. The morphologically good blastocysts were defined by Gardner’s classification 4BB or better [ 10 ], while the morphologically poor blastocysts were those given with C grade in either trophectoderm or inner cell mass (ICM). A written informed consent was obtained from all participating patients regarding PGT-A and euploid or low-frequency mosaic (LFM) blastocyst transfer, in accordance with the corresponding approval by the Institutional Reviewer Board (19 − 006). All treatment cycles used autologous oocytes, and no donor oocyte were included. All participating patients had single euploid blastocyst transfer within one year from the date of the PGT-A report. Additional written informed consent was obtained from all participants to perform thawed BT with HETM, in accordance with the corresponding approval by the Institutional Reviewer Board of Sugiyama Clinic for retrospective investigation of unidentified data (21 − 006). All participants received vaginal ultrasound imaging, hysterosalpingography, and hysteroscopy, to exclude uterine abnormality, uterine myoma, endometrial polyps, and intrauterine adhesion before participating in the study. No participant showed glucose intolerance and thyroid dysfunction. Exclusion criteria included autoimmune disease, anti-phospholipid syndrome, and chronic endometritis. Ovarian Stimulation, IVF and Embryo culture All patients received ovarian stimulation for ART treatment, following the previously described protocol (11). Daily administration of 50–100 mg of clomiphene citrate (Clomid®, Fuji Pharm, Tokyo) or 2.5-5.0mg of letrozole (Letrozol®, Sawai, Osaka) was started from the 2nd or 3rd day of the menstrual cycle or withdrawal bleeding for 7 days, and 200–300 mg of recombinant follicle-stimulating hormone (rec-FSH; Gonal-F®; Merck BioPharma, Tokyo) or highly purified human menopausal gonadotropin (hMG, HMG-Ferring®, Ferring Pharmaceuticals. Tokyo) was administrated every two days from the 3rd day of the menstrual cycle. On the 10th day of the same cycle, transvaginal ultrasound imaging and hormone analysis was performed to evaluate follicle growth. When the dominant follicles reached at ≥ 20 mm in diameter, the patient was given a recombinant human chorionic gonadotropin (rec-hCG; Ovidrel®; Merck BioPharma, Tokyo) or gonadotropin-releasing hormone (GnRH) agonist or both as the maturation trigger [ 12 ]. Approximately half of the patients were stimulated with this protocol, while the other half were treated with a modified progestins-primed ovarian stimulation protocol (modified PPOS), which differed only in the administration of progestins (10 mg of medroxyprogesterone Acetate; Hysron®, Kyowo-Kirin, Tokyo) from 8th to the day of maturation trigger. Thirty-five to thirty-six hours after the maturation trigger, the patient underwent transvaginal oocyte retrieval with or without general or local anesthesia. The retrieved oocytes were inseminated by either conventional insemination or intracytoplasmic sperm injection (ICSI), depending on the diagnosis after sperm preparation or the patients’ characteristics. Piezo-ICSI method was used for all ICSI cases [ 13 ]. After confirmation of normal fertilization, the fertilized oocytes were continuously cultured to the blastocyst stage in the time-lapse incubator (Geri™, Genea Biomedx, VIC, Austlaria) supplied with mixed gas (4% O 2 , 6% CO 2 , and 89% N 2 ) [ 14 ]. All embryos were cultured individually in GEMS GERI MEDIUM™ (Genea Biomedx, VIC, Australia), from immediately after insemination until trophectoderm (TE) biopsy, when the blastocysts had reached 4BB or better in Gardner’s classification [ 10 ]. TE biopsy, NGS, and blastocyst cryopreservation TE biopsy samples were isolated in our laboratory by mechanical dissection technique without laser assisted hatching (LAH). Under continuous time-lapse monitoring, the expanded blastocysts with adequate number of TE cells were biopsied for PGT-A. First, a small hole was made in zona pellucida of the embryo using infrared diode laser (Saturn 5™ Active, Cooper Surgical, CT, USA), and blastocoele collapse was induced. Next, a biopsy pipette (Kitazato, Shizuoka, Japan) was inserted through that small hole into the perivitelline space to aspirate TE cells. After TE aspiration, approximately 5–10 TE cells were excised by mechanical blunt dissection technique [ 15 ]. These procedures were performed with micromanipulation instruments while keeping the embryo droplets of PGD biopsy medium (Global, LifeGlobal, USA). The isolated TE cells were washed twice in sterile phosphate-buffered saline (PBS) supplemented with 1% polyvinylpyrrolidone (PVP), then transferred into a 0.2 ml PCR tube with 2.5 µL droplet of PBS and kept immediately in -20°C until the DNA analysis. All biopsy samples were sent to the laboratory of KITAZATO BIOLABORATORY, Tokyo for the whole genome amplification (WGA) by Sureplex DNA Amplification System (Illumina, CA, USA), then analyzed using next generation sequencing (NGS) in Miseq System (Illumina, CA, USA). Following the NGS run, the CNV chart was generated by BlueFuse Multi Software (Illumina, CA, USA) [ 7 ]. Following biopsy, all remaining blastocysts were cryopreserved using the vitrification method. [ 16 , 17 ], in readiness for the PGT-A analysis reports and the subsequently ploidy status assessments (euploid, mosaic, and aneuploid). Those blastocysts showing less than or equal to 20% of mosaicism or only segmental mosaicism were called as euploid, thus considered acceptable for thawed BT [ 9 ]. Thawed euploid blastocyst transfer cycle with or without HETM Prior to thawed-BT with an euploid blastocyst, the patient underwent uterine endometrial preparation for either natural ovulatory cycle (NOC) or hormone replacement cycle (HRC) depending on the pattern of patients’ menstrual cycle. For patients undergoing the NOC protocol, the blastocyst transfer was performed five days after ovulation, which was verified by both vaginal ultrasonography and the elevation of serum progesterone level (≥ 2ng/ml). Dydrogesterone tablets (Duphaston®, Mylan EPD, Tokyo) were administrated at 30 mg per a day as luteal support, starting from the day after ovulation until the day of pregnancy test, and 125 mg of hydroxyprogesterone caproate (Progeston depot®; Fuji pharma, Tokyo) was injected once on the day of embryo transfer. For patients undergoing the HRC protocol, the uterine endometrium was prepared using both 1.25 mg of conjugated estrogen tablets (Premarin 0.625 mg®, Wyeth, Tokyo, Japan) and 2.88 mg transdermal estradiol patch (Estrana TAPE 0.72 mg®, Hisamitsu Pharmaceutical, Tokyo, Japan), started on the third day of the menstrual cycle or three days after withdrawal bleeding until the day of pregnancy test. 90 mg vaginal progesterone gel (OneCrinone®; Merck BioPharma, Tokyo) and 30mg/day of dydrogesterone tablets were administered, starting from day 13 of menstrual cycle. The thawed BT was performed five days after commencement of progesterone treatment [ 5 ]. Transdermal estradiol patch, dydrogesterone tablets, and vaginal progesterone gel were continued until the day of pregnancy test, and after positive pregnancy test was verified, the patient maintained the same treatment protocol until 9–10 weeks of gestation. We prepared the embryo transfer dish the day before thawed BT, in which 1.0 ml of the embryo transfer medium was taken to the transfer dish and equilibrated for more than 18 hours in an incubator set at 37°C supplied with a mixed gas of 6% CO2, 5% O2 and 89% N2. EmbryoGlue® (Vitrolife, Sweden) was used for the HETM group while continuous single culture medium (CSC-C, Fuji Film, Japan) was used for the control group without additional hyaluronan. On the day of thawed BT, the cryopreserved euploid blastocysts were warmed and checked for the viability. Some of those embryos verified alive had a small hole created in the zonae pellucida by LAH (Saturn 5 TM ACTIVE; Cooper Surgical) before recovery culture for several hours. Blastocysts were assessed again for the morphological grades using Gardner’s Classification [ 10 ] just prior to transfer. Blastocysts which remained a 4BB or better after the thawing process were marked as morphologically good blastocysts, while those blastocysts with C grade in either trophectoderm or inner cell mass (ICM) were marked as morphologically poor blastocysts. The euploid blastocyst selected for transfer were placed in the transfer dish, prepared with equilibrated HETM or control medium, and incubated for a maximum of one-half hours. The euploid blastocyst were replaced into the patient’s uterus transcervically using a soft catheter (Kitazato ET catheter, Kitazato Supply, Shizuoka, Japan) under transvaginal ultrasonography. A single selected blastocyst was replaced into the patient’s uterus transcervically using a soft catheter (Kitazato ET catheter, Kitazato Supply, Shizuoka, Japan) while being monitored under transvaginal ultrasonography. Clinical outcomes The pregnancy test was performed 9 days after the thawed BT by measuring serum human chorionic gonadotropin (hCG), where the positive pregnancy test was defined as more than 10 IU/ml of serum hCG. A clinical pregnancy was recognized when the development of a gestational sac (GS) was verified by transvaginal ultrasound imaging 16–21 days after the thawed ET. A miscarriage was defined when the fetal heart movement was unable to be confirmed by transvaginal ultrasound imaging in 9 weeks after gestation. The clinical pregnancy rate (CPR) was calculated from the number of cycles with confirmed GS and the total cycle number of thawed BT. The miscarriage rate (MR) was calculated from the number of the miscarriage cycles and the total number of the clinical pregnancies. Statistical analysis The data acquired in the clinic was statistically analyzed by Fisher’s exact test or non-parametric test. A multivariable logistic regression model was constructed in order to find independent risk factors and complications associated with the pregnancy outcomes after thawed BT using morphologically poor euploid blastocyst, while controlling for confounders. Odds ratios (OR) and their 95% confidence interval (CI) were computed. A p-value of < .05 was considered statistically significant. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics [ 18 ]. Results Patient’s characteristics shown in the Table 1 . The median age of the control and HETM groups were both 39.0 with no significant difference (p = .72). The proportions of patients in advanced maternal age (≥ 40 years old) in the control and HETM use groups were 40.4% and 42.1%, respectively, without significant difference (p = 1). The proportion of RIF patients in the HETM group was 50.0%, which was significantly higher than the control group (25.5%, p = .025), while the proportions of PRL patients were comparable (p = .447). The median number of previous ET attempts in the control and HETM groups were 1 and 2, respectively, but the difference was not statistically significant (p=. 22). Conventional insemination method was used less frequently than ICSI (31.8% and 68.2%, respectively, p = .065), but we considered the difference was not significant enough to influence the overall outcomes. In the endometrial preparation before embryo transfer, the NOC protocol was selected for approximately half of the participants (56.5%), while the HRC protocol was selected for another half (43.5%) of the participants (p = .66). The proportions of patients underwent LAH after thawing was also similar in these two groups (29.8% in the control and 28.9% in the HETM groups, respectively, p = 1.0) Table 1 The backgrounds of the morphologically poor euploid blastocyst transfer with or without HETM. Control HETM P value Number of thawed BT 47 38 Age, years* [IQR**] 39.0 [37.0–40.0] 39.0 [34.0-40.8] 0.72 Proportion of AMA # (≥ 40), n (%) 19 (40.4) 16 (42.1) 1.0 Proportion of RIF ## , n (%) 12 (25.5) 19 (50.0) 0.025 Proportion of PRL ### , n (%) 0 (0) 1 (2.6) 0.447 Previous ET attempts, n* [range, times] 1 [0–3] 2 [0–5] 0.22 Mode of insemination (IVF/ICSI), n 19/28 8/30 0.065 Endometrial preparation (NOC/HRC) ǂ , n 28/19 20/18 0.66 Proportion of LAH ǂǂ , n (%) 14 (29.8) 11 (28.9) 1.0 *median, **IQR; interquartile range # AMA; advanced maternal age, ## RIF; repeated implantation failure, ### RPL; recurrent pregnancy loss ǂ NOC/HRC; natural ovulatory cycle/hormone replacement cycle, ǂǂ LAH; laser assisted hatching The clinical pregnancy rate (CPR) from transfer of a morphologically poor euploid blastocyst was 32.9% (28/85), and it was significantly lower than the CPR from transfer of a morphologically good blastocysts reported by Sato et al. [ 19 ]. Among the thawed-BT with morphologically poor blastocysts, we found significant improvement in the CPR of HETM group compared to the control group (47.4% [18/38] and 21.3% [10/47], P = .019), while we did not see any differences in CPRs by the use of LAH (LAH+; 36.0% [9/25] and LAH-; 31.6% [19/60], respectively, p = 0.89). (Fig. 2 ). These results led us to identify the predictive factors for achieving a higher chance of pregnancy from the embryo transfer with morphologically poor euploid blastocyst, from a comparison between the group of 28 pregnant patients against the group of 57 non-pregnant patients (Table 2 ). The average age in the pregnant group was slightly younger than the non-pregnant group (37.5 and 39.0 years, respectively, p = .242). There were no significant differences found in either the proportions of advanced maternal age, RIF, PRL, the number of previous ET attempts, or the insemination method. Comparing the endometrial preparation protocols used before thawed BT, the NOC protocol was used more frequently than the HRC protocol (56.4% and 43.6%, respectively). The NOC protocol was used in 67.9% of the pregnant group, which was higher than the non-pregnant group (50.9%, p = .76). The proportion of HRC protocol in the pregnant and non-pregnant groups were 32.1% and 49.1%, respectively, and the difference was not significant (p = .12). The proportion of embryos underwent LAH method were also comparable between the pregnant and non-pregnant groups (32.15% and 28.1%, respectively, p = .801). Interestingly, the proportion of embryos prepared in the HETM medium was 64.3% in the pregnant group, which was significantly higher than the non-pregnant group (35.1%, p = .019). Table 2 Predictive factors for achieving pregnancy for the morphologically poor euploid blastocyst Pregnant group n = 28 Non-pregnant group n = 57 P-value Univariate analysis OR (95%CI) Multivariate analysis OR (95%CI) Age, years* [IQR**] 37.5 [34.0–40.0] 39.0 [37.0–40.0] 0.242 0.94 (0.83–1.07) 0.96 (0.77–1.20) Proportion of AMA # (≥ 40), n (%) 10 (35.7) 25 (43.9) 0.494 0.71 (0.28–1.81) 0.94 (0.19–4.60) Proportion of RIF ## , n (%) 8 (28.6) 23 (40.4) 0.343 0.59 (0.22–1.57) 1.02 (0.200–5.14) Proportion of PRL ### , n (%) 7 (25.0) 8 (14.0) 0.237 2.04 (0.66–6.34) 2.53 (0.672–9.53) Previous ET attempts, n [range, times] 0.5 [0.0–4.0] 2.0 [0.0–4.0] 0.087 0.85 (0.71–1.03) 0.83 (0.620–1.10) Mode of insemination (IVF/ICSI), n 8/20 19/38 0.805 1.25 (0.47–3.36) 0.87 (0.27–2.86) Endometrial preparation (NOC†/HRC††), n (%) 19/38 (32.1/67.9) 29/28 (50.9/49.1) 0.167 0.49 (0.19–1.27) 0.47 (0.15–1.43) Proportion of LAH ǂ , n (%) 9 (32.1) 16 (28.1) 0.801 1.21 (0.46–3.24) 1.59 (0.50–5.08) HETM ǂǂ , n (%) 18 (64.3) 20 (35.1) 0.019 3.33 (1.29–8.57) 5.08 (1.62-16.0) *mean, **IQR; interquartile range # AMA; advanced maternal age, ## RIF; repeated implantation failures, ### RPL; recurrent pregnancy losses †NOC; natural ovulatory cycle, †† HRC; hormone replacement cycle, ǂ LAH; laser assisted hatching, ǂǂ HETM; hyaluronan-enriched transfer medium† The result from univariate analysis indicated that the use of HETM before embryo transfer increased the chance of positive pregnancy outcomes (OR = 3.33, 95% CI = 1.29–8.57). The multiple logistic regression analysis found that the use of HETM is a predictive factor of positive pregnancy outcomes (OR = 5.08, 95% CI = 1.62-16.0). Discussion The most common way of practice in ART labs to select embryos for blastocyst transfer is based on the morphological evaluation by Gardner’s grading method [ 10 ]. Timelapse image acquisition systems opened the way to propose alternative methods to select blastocysts for transfer using annotation parameters such as time to reach blastocyst stage, blastocyst expansion speed, ICM and TE grades [ 10 ]. In addition to embryo selection methods based on morphology, the increasing use of PGT-A allows clinicians to select the blastocysts with higher probability of implantation based on the chromosomal profiles of embryo, or euploidy [ 19 , 20 ]. The potential benefits of combining embryo morphology and PGT-A have been shown in recent publications, that morphological grades could still differentiate the chance of clinical and ongoing pregnancy among euploid embryos [ 20 , 21 ]. Our data supports these findings: the clinical pregnancy rate from the transfer of morphologically poor euploid blastocysts was 32.9% (28/85), while the clinical pregnancy rate of morphological good blastocyst was 62.2% (729/1,172, data not shown in the table). Our result also demonstrated that HETM group in thawed-BT of morphologically poor euploid blastocysts resulted in much higher success rates than the control group in the same setting. Patient demographics including median age, the proportion of advanced maternal age, and the proportion of PRL, did not correlate with then difference found between these two groups, suggesting that the use of HETM by itself could improve the outcome for morphological poor blastocysts. The proportion of ICSI cycles was slightly higher in the HETM group but the difference was not significant enough. Our recent report found potentially profound impacts of endometrial preparation method to the pregnancy success rate of euploid blastocyst transfer, showing the HRC protocol being a negative predictive factor for clinical pregnancy [ 8 ]. On the contrary, the proportions of NOC and HRC protocols were quite similar between the control and HETM groups in this study, and there was no bias found regarding the endometrial preparation protocols before embryo transfer. Instead, this study showed that the use of HETM in the preparation of blastocyst before transfer improved the clinical pregnancy rate of morphologically poor euploid blastocyst (47.4% vs. 21.3%, p = .0194), indicating that hyaluronan supplementation could help morphologically poor euploid blastocysts implant. Hyaluronan is one of the essential molecules found in key reproductive events including ovulation, fertilization, embryogenesis, implantation and trophoblast invasion [ 22 – 24 ]. Hyaluronan is synthesized as a liner polymer structure of glycosaminoglycan which consists of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine, and it was formed in polymer of various molecular sizes up to 10 7 Da. It is produced predominantly by three isoforms of hyaluronic acid synthase (HAS1-3), each generating polymers of different lengths. Turnover of hyaluronan molecules in physiological environments is relatively short, either by oxidation or enzymatic degradation by a family of hyaluronidases (HYALs) [ 25 ]. Among three main isoforms of HYALs, hyaluronidase 2 (HYAL2) is expressed on the cell surface with a glycosylphosphatidylinositol (GPI)-anchor attached to the extracellular side of the plasma membrane [ 25 , 26 ]. Biological functions of hyaluronan polymers in extracellular matrix are modulated through interaction with a variety of hyaluronan-binding proteins, including the most notable hyaluronan-receptor, CD44 [ 24 , 25 , 27 ]. HYAL-2 is involved in the processing of long chain hyaluronan in the extracellular space through the interaction with CD44, and the cleavage of hyaluronan via HYAL-2/CD44 complex is essential to the subsequent receptor-mediated internalization of hyaluronan into the cytoplasm [ 28 ]. Kong et al. recently demonstrated that successful transition of uterine endometrium for establishment of pregnancy requires selective elimination of pro-inflammatory senescent decidual cells by uterine natural killer cells [ 29 ]. They also reported that morphologically poor human blastocysts derived from IVF/ICSI express lower levels of HYAL2 compared to morphologically good blastocysts (Gardner’s classification; ≥BB, p = .003). Moreover, the successfully implanted blastocysts had higher level of HYAL2 expression compared to the blastocysts failed to implant after transfer (p = .042). This suggests that the morphologically poor euploid blastocysts in our study also have lower levels of HYAL2 on the cell structure, which could limit the interaction with CD44 to process and internalize extracellular hyaluronan [ 26 , 29 ]. Although the information of average molecular weight of hyaluronan in EmbryoGlue® is not available, the enriched-hyaluronan in this transfer medium might help facilitate the CD44-mediated internalization process to improve of the implantation of morphologically poor euploid blastocysts. An increase of high molecule weight hyaluronan would also increase the density of extracellular matrix to a level closer to uterine endometrium, which could give rise to an immunotolerant reaction via uterine natural-killer cell [ 29 ]. These series of factors could confer additive effects to help the implantation of morphologically poor blastocysts. As has been demonstrated by numerous reports and publications, morphologically poor blastocysts often resulted in lower implantation rates, even when only the euploid embryos are selected for transfer. Our study strongly suggested that HETM used at the time of blastocyst transfer can significantly improve the likelihood of morphologically poor euploid blastocysts to achieve clinical pregnancy. This provides a relatively simple yet powerful tool for clinicians and embryologists when they find the morphology of blastocysts has worsened after freeze-and-thaw. Our own experience as well as previously published data strongly suggested that the concentration of hyaluronan, and possibly the chain length of hyaluronan polymers, could be essential factors to consider in selecting embryo transfer medium most suitable to improve the chance of clinical pregnancy. It led us to use EmbryoGlue® as HETM in our study as the hyaluronan concentration data is disclosed by the manufacturer (0.5mg/ml). The data shown in this study supports our previous hypothesis, that HETM is especially helpful to improve the success rates of those cycles associated with less favorable factors, either in patient characteristics or the embryos to be transferred. Declarations Acknowledgements The authors would like to thank Kathryn Gebhardt for English editing for this paper. Conflict of interest Koji Nakagawa, Takashi Horikawa, Yuji Orita, Emi Yamashiro, Hideaki Watanabe, Asako Shirai, Hisayo Kataoka, Keiji Kuroda, Satoru Takamizawa, Rikikazu Sugiyama declare that they have no conflicts of interest. Souichi Ogata in an employee of Vitrolife K.K. Funding This study was conducted with the own funding of author’s group without any external source. Authors contributions Nakagawa K is a principal investigator. Horikawa T, Kataoka H, Kuroda K, and Takamizawa, contributed to collect clinical data, and Yamashiro E, Watanabe E, Watanabe H, and Shirai A contributed to deal with ART procedure and handling embryos. Orita Y performed statistically data analysis. Ogata S is the supervisor of this study. Sugiyama R is organized this study. All of them agree with the content of this manuscript. Everyone in the author list reviewed the final manuscript before the submission. Human Rights Statements and informed consent The study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards, and it was reviewed and approved by the institutional review board of Sugiyama Clinic. All patients have received and signed on an informed written consent form before entering the study, and they were also informed for an option to abort from the study at any time during the treatment. 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Nakagawa K, Takahashi C, Nishi Y, Juen H, Sugiyama R, Kuribayahsi Y, Sugiyama R. Hyaluronan-enriched transfer medium improves outcome in patients with multiple embryo transfer failures. J Assist Reprod Genet. 2012: 29: 679-85. Heymann D, vidal L, Or Y, Shoham Z. Hyaluronic acid in embryo transfer media for assisted reproductive technologies. Cochrane Database of Systematic Reviews 2020, CD007421. DOI: 10.1002/14651858. Penzias A, Bendikson K, Butts S, Coutifaris C, Falcone T, Fossum G, Gitlin S, Gracia Hansen K, La Barbera A, Mersereau J, Odem R, Paulson R, Pfeifer S, Pisarska M, Rebar R, Reindollar R, Rosen M, Sandlow J, Vernon M, Widra E. The use of preimplantation genetic testing for aneuploidy (PGT-A): a committee opinion. Fertil Steril. 2018; 109:429–36. Horikawa T, Nakagawa K, Tonouchi E, Kuroda K, Watanabe H, Takamizawa S, Sugiyama R. Trophectoderm grade and the day of TE biopsy significantly corelate with pregnancy rate in euploid blastocyst transfer. Fertil Steril 2021; 116: e247 PGDIS. PGDIS Position Statement on Chromosome Mosaicism and Preimplantation Aneuploidy Testing at the Blastocyst Stage. 2016;1–2. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril. 2000; 73: 1155-8. Sugiyama R, Nakagawa K, Nishi Y, Ojiro Y, Juen H, Sugiyama R, Kuribayashi Y. Using a mild stimulation protocol combined with clomiphene citrate and recombinant follicle-stimulating hormone to determine the optimal number of oocytes needed to achieve pregnancy and reduce the concerns of patients. Reprod Med Biol. 2013; 12:105–10. Nakagawa K, Oba M, Ehara K, Ishigaki N, Ino N, Itakura A, Ysutsumi R, Nakao K, Ojiro Y, Sugiyama R. Clinical outcomes of assisted reproductive technology treatment by using a self-injection of recombinant human chorionic gonadotropin as the final maturation trigger. Reprod Med Biol. 2018; 17: 203–8. Hiraoka K, Kitamura S. Clinical efficiency of Piezo-ICSI using micropipettes with a wall thickness of 0.625 μm. J Assist Reprod Genet. 2015; 32: 1827–33. Nakagawa K, Shirai A, Nishi Y, Sugiyama R, Kuribayashi Y, Sugiyama R, Inoue M. A study of the effect of an extremely low oxygen concentration on the development of human embryos in assisted reproductive technology. Reprod Med Biol. 2010; 9: 163-8. Yang D, Feng D, Gao Y, Sagnelli M, Wang X, Li D. An effective method for trophectoderm biopsy using mechanical blunt dissection: a step-by-step demonstration. Fertil Steril. 2020; 114: 438–439. Sugiyama R, Nakagawa K, Shirai A, Sugiyama R, Nishi Y, Kuribayashi Y, Inoue M. Clinical outcomes resulting from the transfer of vitrified human embryos using a new device for cryopreservation (plastic glade). J Assist Reprod Genet. 2010; 27: 161-7 Practice Committee of the American Society for Reproductive Medicine. A review of best practices of rapid-cooling vitrification for oocytes and embryos: a committee opinion. Fertil Steril. 2021; 115: 305–10. Kanda Y. Investigation of the freely available easy-to-use software “EZR” for medical statistics. Bone Marrow Transplantation. 2013; 48: 452–8. Sato T, Sugiura-Ogasawara M, Ozawa F, Yamamoto T, Kato T, Kurahashi H, Kuroda T, Aoyama N, Kato K, Kobayashi R, Fukuda A, Utsunomiya T, Kuwahara A, Saito H, Irahara M. Preimplantation genetic testing for aneuploidy: a comparison of live birth rates in patients with recurrent pregnancy loss due to embryonic aneuploidy or recurrent implantation failure. Hum Reprod. 2019; 34: 2340-8. Irani M, Reichman D, Robles A, Melnick A, Davis O, Zaninovic N, Xu K, Rosenwaks Z. Morphologic grading of euploid blastocysts influences implantation and ongoing pregnancy rates. Fertil Steril. 2017; 107: 664-70. Gonzalez XV, Odia R, Naja R, Serhal P, Saab W, Seshadri S, Ben-Nagi J. Euploid blastocysts implant irrespective of their morphology after NGS-(PGT-A) testing in advanced maternal age patients. J Assist Reprod Gent. 2019; 36: 1623-9. Fouladi-Nashta AA, Raheem KA, Marei WF, Ghafari F, Hartshorne GM. Regulation and roles of the hyaluronan system in mammalian reproduction. Reproduction. 2017; 153: R43-R58, doi:10.1530/REP-16-0240. Ruane PT, Buck CJ, Babbington PA, Aboussahoud W, Berneau SC, Wwetwood M, Kimber SJ, Aplin JD, Brison DR. The effects of hyaluronate-containing medium on human embryo attachment to endometrial epithelial cells in vitro. Hum Reprod Open. 2020; hoz033, doi:10.1093/hropen/hoz03. Takahashi H, Takizawa T, Matsubara S, ohkuchi A, Kuwata T, Usui R, Matsumoto H, Sato Y, Fujiwara H, Okamoto A, Suzuki M, Takizawa T. Extravillous trophoblast cell invasion is promoted by the CD44- hyaluronic acid interaction. Placenta. 2014; 35:163-170. Dicker KT, Gurski LA, Pradhan-Bhatt S, Witt RL, Farach-Carson MC, Jia X. Hyluronan: A Simple Polysaccharide with Diverse Biological Functions. Acta Biomater. 2014; 10: 1558-70. Rai SK, Duh FM, Vigdorovich V, Danilkovitch-Miagkova A, Lerman MI, Miller AD. Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation. Proc. Natl. Acad. Sci. U S A. 2001; 98:4443–4448. Yang B, Yang BL, Savani RC, Turley EA. Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein. EMBO J. 1994, 13:286–296. Harada H. and Takahashi M. CD44-dependent intracellular and extracellular catabolism of hyaluronic acid by hyaluronidase-1 and -2. J. Biol. Chem. 2007, 282(8): 5597-5607. Kong CS, Ordoñez AA, Turner S, Tremaine T, Muter J, Lucas ES, Salisbury E, Vassena R, Fouladi-Nashta AA, Tiscornia G, Hartshorne G, Brosens JJ, Brighton PJ. Involvement of uterine natural killer cells in the natural selection of human embryos at implantation. FASEB J. 2021, 35 (4): e21336. Cite Share Download PDF Status: Published Journal Publication published 31 May, 2023 Read the published version in Archives of Gynecology and Obstetrics → Version 1 posted Reviewers agreed at journal 19 Jan, 2023 Reviewers invited by journal 11 Jan, 2023 Editor invited by journal 11 Jan, 2023 Editor assigned by journal 30 Dec, 2022 First submitted to journal 29 Dec, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-2427879\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":166906168,\"identity\":\"03df8661-7215-4ed6-87d4-c39481826f18\",\"order_by\":0,\"name\":\"KOJI NAKAGAWA\",\"email\":\"data:image/png;base64,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\",\"orcid\":\"https://orcid.org/0000-0003-0874-5894\",\"institution\":\"Sugiyama Clinic\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"KOJI\",\"middleName\":\"\",\"lastName\":\"NAKAGAWA\",\"suffix\":\"\"},{\"id\":166906169,\"identity\":\"8317425b-b6ec-43c7-94d7-210cea049e48\",\"order_by\":1,\"name\":\"Takashi Horikawa\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Takashi\",\"middleName\":\"\",\"lastName\":\"Horikawa\",\"suffix\":\"\"},{\"id\":166906170,\"identity\":\"050c8bdb-2f7e-4f36-be75-9bc62dbad266\",\"order_by\":2,\"name\":\"Yuji Orita\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yuji\",\"middleName\":\"\",\"lastName\":\"Orita\",\"suffix\":\"\"},{\"id\":166906171,\"identity\":\"ea4fa993-b646-40f7-a23f-8b71752c2762\",\"order_by\":3,\"name\":\"Emi Yamashiro\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Emi\",\"middleName\":\"\",\"lastName\":\"Yamashiro\",\"suffix\":\"\"},{\"id\":166906172,\"identity\":\"c7e25312-5da9-4a32-8ece-74665705a493\",\"order_by\":4,\"name\":\"Hideaki Watanabe\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hideaki\",\"middleName\":\"\",\"lastName\":\"Watanabe\",\"suffix\":\"\"},{\"id\":166906173,\"identity\":\"623410dc-a6be-44f1-b1f1-962ee953174d\",\"order_by\":5,\"name\":\"Asako Shirai\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Asako\",\"middleName\":\"\",\"lastName\":\"Shirai\",\"suffix\":\"\"},{\"id\":166906174,\"identity\":\"15f016d8-10ef-458e-9434-e4321c7143f0\",\"order_by\":6,\"name\":\"Souichi Ogata\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Souichi\",\"middleName\":\"\",\"lastName\":\"Ogata\",\"suffix\":\"\"},{\"id\":166906175,\"identity\":\"5d4e2802-9613-4a5d-8a97-05ae52b75610\",\"order_by\":7,\"name\":\"Hisayo Kataoka\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hisayo\",\"middleName\":\"\",\"lastName\":\"Kataoka\",\"suffix\":\"\"},{\"id\":166906176,\"identity\":\"03f2ad0d-da63-4b37-baf8-54886dc849d0\",\"order_by\":8,\"name\":\"Keiji Kuroda\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Keiji\",\"middleName\":\"\",\"lastName\":\"Kuroda\",\"suffix\":\"\"},{\"id\":166906177,\"identity\":\"991cea6f-a72e-424d-b7ac-911e7f416307\",\"order_by\":9,\"name\":\"Satoru Takamizawa\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Satoru\",\"middleName\":\"\",\"lastName\":\"Takamizawa\",\"suffix\":\"\"},{\"id\":166906178,\"identity\":\"b1418310-ff7a-44ae-b1d8-9bce25b0acc8\",\"order_by\":10,\"name\":\"Rikikazu Sugiyama\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Rikikazu\",\"middleName\":\"\",\"lastName\":\"Sugiyama\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2022-12-30 09:17:12\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-2427879/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-2427879/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1007/s00404-023-07083-9\",\"type\":\"published\",\"date\":\"2023-05-31T21:03:22+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":31559099,\"identity\":\"03ce1226-fa21-4dc2-ac3e-b5dc3b1fead5\",\"added_by\":\"auto\",\"created_at\":\"2023-01-13 21:02:16\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":23833,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe selection flowchart of thawed-BT cycles included in this study. Of 1,168 thawed blastocyst transfer after PGT-A, 129 thawed BTs were performed with mosaic blastocysts, while the remaining 1,039 thawed BTs were performed with euploid blastocysts. All transferred embryos had been subjected to morphological assessments after thawing, which resulted in 954 blastocysts with good morphological grades and 85 blastocysts with poor morphological grades available to transfer. In the thawed BT with these 85 morphologically poor euploid blastocysts, 47 of these were prepared with HETM just before the transfer, while the remaining 38 were prepared with the control medium.\\u003c/p\\u003e\\n\\u003cp\\u003eBT; blastocyst transfer, PGT-A; preimplantation genetic testing for aneuploidy, HETM; hyaluronan-enriched transfer medium\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig1Glue.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2427879/v1/41502f3fbc02bb270c1b6085.png\"},{\"id\":31559100,\"identity\":\"b8445a4f-3447-4ef1-b8e0-f2d1cea3ccb7\",\"added_by\":\"auto\",\"created_at\":\"2023-01-13 21:02:16\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":29076,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThis graph indicates clinical pregnancy rates of the morphologically poor euploid blastocysts prepared with different conditions: HETM or control media, and the usage of LAH after thawing the embryos. The CPR of the HETM group was 47.4% [18/38] and significantly higher than that of the control group (21.3% [10/47], P=.019). In contrast, LAH did not influence the CPRs in the same settings (LAH+; 36.0% [9/25] and LAH-; 31.6% [19/60], p=0.89).\\u003c/p\\u003e\\n\\u003cp\\u003eHETM; hyaluronan-enriched transfer medium , LAH; laser assisted hatching\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2427879/v1/790de7a3c038ae0c25c74930.png\"},{\"id\":44732172,\"identity\":\"d037aadb-d0b8-446a-ae61-5315b9fd0cc6\",\"added_by\":\"auto\",\"created_at\":\"2023-10-16 21:53:41\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":416602,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2427879/v1/d6c25f4f-f794-4e4c-9d38-c4abefd6dcf1.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Hyaluronan-enriched transfer medium (HETM) can improve the implantation rate in morphologically poor euploid blastocyst transfer\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eModern assisted reproductive technology (ART) treatments consist of various processes, starting from ovarian stimulation, oocyte pick-up, insemination, embryo culture and finally embryo transfer. Hyaluronan-enriched transfer medium (HETM) has been clinically used in many ART clinics as a medium specially formulated for embryo transfer, which was first reported in 2002 to improve the implantation rates of human embryos in ART treatment [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e], followed by many other RCT studies demonstrating the efficacy of HETM in human embryo transfer [\\u003cspan additionalcitationids=\\\"CR3\\\" citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Reported by Urman et al., HETM was effective in improving the implantation rates in a fresh embryo transfer cycle for those patients with history of one or more implantation failures and patients of advanced maternal ages [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Our group has also previously reported that the clinical pregnancy rates of those patients with repeated implantation failures (RIF) was improved in the thawed embryo transfer cycles using HETM [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Having this experience, our group have used HETM as the transfer medium for those patients with applicable histories or indications, or those who made a specific request after having an adequate consultation with the clinicians. More recently, a Cochrane review published in 2020, which cited data reported from a number of previous clinical studies using HETM in embryo transfer, supported the addition of hyaluronic acid (HA) in embryo transfer medium to improve the clinical outcomes of ART treatments, including the improvements in live births per ART trteatments and the decrease the rates of miscarriage [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. Interestingly, we noted that many previous studies had selected embryos with good morphological grades for transfer, because in general poor morphological grades were thought to indicate lower implantation potential.\\u003c/p\\u003e\\u003cp\\u003eMore recently, preimplantation genetic testing for aneuploidy (PGT-A) is becoming commonplace, and it has been widely implemented in the treatment for patients with RIF or recurrent pregnancy losses, to reduce the chance of another failure in embryo transfer. While blastomere biopsy used to be a common procedure for PGT-A, nowadays it has been replaced almost entirely with trophectoderm (TE) biopsy taken at blastocyst stage [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. In Japan, the selection of euploid blastocysts for transfer following PGT-A has been allowed since 2019. Recently, we reported that the morphologically good blastocysts had higher clinical pregnancy rates than morphologically poor blastocysts even when we only selected euploid blastocysts for transfer [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. But there have been seldom clinical reports specially asking how HETM could improve the clinical pregnancy rates of blastocysts with poor morphological grades, or enriched hyaluronan could potentially help the implantation of a morphologically poor blastocyst.\\u003c/p\\u003e\\u003cp\\u003eTherefore, we designed our study to determine whether the use of HETM improves the clinical pregnancy rate (CPR) of patients who underwent euploid blastocyst transfer with poor morphological grade. Selecting only euploid blastocysts by PGT-A, our study specially addresses the relationship between the embryo morphology and the transfer success rates in the presence of high concentration HA in embryo transfer medium.\\u003c/p\\u003e\"},{\"header\":\"Materials And Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eStudy design\\u003c/h2\\u003e\\u003cp\\u003eWe conducted a single center, cross-sectional study between July 2020 and June 2022, which recruited a total of 1,168 thawed blastocyst transfers (thawed BT) at Sugiyama Clinic Shinjuku, Tokyo, Japan. A flowchart of the patient selection process is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. All blastocysts used in this study were called as euploid or mosaic based on PGT-A results. Those blastocysts showing 20% or less of mosaicism or segmental mosaicism were called as euploid and accepted to thawed BT [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. One hundred twenty-nine thawed BT were performed with mosaic blastocysts, while the remaining 1,039 thawed BTs were performed with euploid blastocysts. All transferred embryos had been subjected to morphological assessments after thawing, which resulted in 954 blastocysts with good morphological grades and 85 blastocysts with poor morphological grades available to thawed BTs. Of the 85 morphologically poor euploid blastocysts, 47 of these were used for HETM and the remaining 38 were transferred without HETM at thawed BT. The morphologically good blastocysts were defined by Gardner\\u0026rsquo;s classification 4BB or better [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e], while the morphologically poor blastocysts were those given with C grade in either trophectoderm or inner cell mass (ICM).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e A written informed consent was obtained from all participating patients regarding PGT-A and euploid or low-frequency mosaic (LFM) blastocyst transfer, in accordance with the corresponding approval by the Institutional Reviewer Board (19\\u0026thinsp;\\u0026minus;\\u0026thinsp;006). All treatment cycles used autologous oocytes, and no donor oocyte were included. All participating patients had single euploid blastocyst transfer within one year from the date of the PGT-A report. Additional written informed consent was obtained from all participants to perform thawed BT with HETM, in accordance with the corresponding approval by the Institutional Reviewer Board of Sugiyama Clinic for retrospective investigation of unidentified data (21\\u0026thinsp;\\u0026minus;\\u0026thinsp;006).\\u003c/p\\u003e\\u003cp\\u003eAll participants received vaginal ultrasound imaging, hysterosalpingography, and hysteroscopy, to exclude uterine abnormality, uterine myoma, endometrial polyps, and intrauterine adhesion before participating in the study. No participant showed glucose intolerance and thyroid dysfunction. Exclusion criteria included autoimmune disease, anti-phospholipid syndrome, and chronic endometritis.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eOvarian Stimulation, IVF and Embryo culture\\u003c/h2\\u003e\\u003cp\\u003eAll patients received ovarian stimulation for ART treatment, following the previously described protocol (11). Daily administration of 50\\u0026ndash;100 mg of clomiphene citrate (Clomid\\u0026reg;, Fuji Pharm, Tokyo) or 2.5-5.0mg of letrozole (Letrozol\\u0026reg;, Sawai, Osaka) was started from the 2nd or 3rd day of the menstrual cycle or withdrawal bleeding for 7 days, and 200\\u0026ndash;300 mg of recombinant follicle-stimulating hormone (rec-FSH; Gonal-F\\u0026reg;; Merck BioPharma, Tokyo) or highly purified human menopausal gonadotropin (hMG, HMG-Ferring\\u0026reg;, Ferring Pharmaceuticals. Tokyo) was administrated every two days from the 3rd day of the menstrual cycle. On the 10th day of the same cycle, transvaginal ultrasound imaging and hormone analysis was performed to evaluate follicle growth. When the dominant follicles reached at \\u0026ge;\\u0026thinsp;20 mm in diameter, the patient was given a recombinant human chorionic gonadotropin (rec-hCG; Ovidrel\\u0026reg;; Merck BioPharma, Tokyo) or gonadotropin-releasing hormone (GnRH) agonist or both as the maturation trigger [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. Approximately half of the patients were stimulated with this protocol, while the other half were treated with a modified progestins-primed ovarian stimulation protocol (modified PPOS), which differed only in the administration of progestins (10 mg of medroxyprogesterone Acetate; Hysron\\u0026reg;, Kyowo-Kirin, Tokyo) from 8th to the day of maturation trigger. Thirty-five to thirty-six hours after the maturation trigger, the patient underwent transvaginal oocyte retrieval with or without general or local anesthesia.\\u003c/p\\u003e\\u003cp\\u003eThe retrieved oocytes were inseminated by either conventional insemination or intracytoplasmic sperm injection (ICSI), depending on the diagnosis after sperm preparation or the patients\\u0026rsquo; characteristics. Piezo-ICSI method was used for all ICSI cases [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. After confirmation of normal fertilization, the fertilized oocytes were continuously cultured to the blastocyst stage in the time-lapse incubator (Geri\\u0026trade;, Genea Biomedx, VIC, Austlaria) supplied with mixed gas (4% O\\u003csub\\u003e2\\u003c/sub\\u003e, 6% CO\\u003csub\\u003e2\\u003c/sub\\u003e, and 89% N\\u003csub\\u003e2\\u003c/sub\\u003e) [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. All embryos were cultured individually in GEMS GERI MEDIUM\\u0026trade; (Genea Biomedx, VIC, Australia), from immediately after insemination until trophectoderm (TE) biopsy, when the blastocysts had reached 4BB or better in Gardner\\u0026rsquo;s classification [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e].\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eTE biopsy, NGS, and blastocyst cryopreservation\\u003c/h2\\u003e\\u003cp\\u003eTE biopsy samples were isolated in our laboratory by mechanical dissection technique without laser assisted hatching (LAH). Under continuous time-lapse monitoring, the expanded blastocysts with adequate number of TE cells were biopsied for PGT-A. First, a small hole was made in zona pellucida of the embryo using infrared diode laser (Saturn 5\\u0026trade; Active, Cooper Surgical, CT, USA), and blastocoele collapse was induced. Next, a biopsy pipette (Kitazato, Shizuoka, Japan) was inserted through that small hole into the perivitelline space to aspirate TE cells. After TE aspiration, approximately 5\\u0026ndash;10 TE cells were excised by mechanical blunt dissection technique [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. These procedures were performed with micromanipulation instruments while keeping the embryo droplets of PGD biopsy medium (Global, LifeGlobal, USA). The isolated TE cells were washed twice in sterile phosphate-buffered saline (PBS) supplemented with 1% polyvinylpyrrolidone (PVP), then transferred into a 0.2 ml PCR tube with 2.5 \\u0026micro;L droplet of PBS and kept immediately in -20\\u0026deg;C until the DNA analysis. All biopsy samples were sent to the laboratory of KITAZATO BIOLABORATORY, Tokyo for the whole genome amplification (WGA) by Sureplex DNA Amplification System (Illumina, CA, USA), then analyzed using next generation sequencing (NGS) in Miseq System (Illumina, CA, USA). Following the NGS run, the CNV chart was generated by BlueFuse Multi Software (Illumina, CA, USA) [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. Following biopsy, all remaining blastocysts were cryopreserved using the vitrification method. [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e], in readiness for the PGT-A analysis reports and the subsequently ploidy status assessments (euploid, mosaic, and aneuploid). Those blastocysts showing less than or equal to 20% of mosaicism or only segmental mosaicism were called as euploid, thus considered acceptable for thawed BT [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e].\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eThawed euploid blastocyst transfer cycle with or without HETM\\u003c/h2\\u003e\\u003cp\\u003ePrior to thawed-BT with an euploid blastocyst, the patient underwent uterine endometrial preparation for either natural ovulatory cycle (NOC) or hormone replacement cycle (HRC) depending on the pattern of patients\\u0026rsquo; menstrual cycle. For patients undergoing the NOC protocol, the blastocyst transfer was performed five days after ovulation, which was verified by both vaginal ultrasonography and the elevation of serum progesterone level (\\u0026ge;\\u0026thinsp;2ng/ml). Dydrogesterone tablets (Duphaston\\u0026reg;, Mylan EPD, Tokyo) were administrated at 30 mg per a day as luteal support, starting from the day after ovulation until the day of pregnancy test, and 125 mg of hydroxyprogesterone caproate (Progeston depot\\u0026reg;; Fuji pharma, Tokyo) was injected once on the day of embryo transfer. For patients undergoing the HRC protocol, the uterine endometrium was prepared using both 1.25 mg of conjugated estrogen tablets (Premarin 0.625 mg\\u0026reg;, Wyeth, Tokyo, Japan) and 2.88 mg transdermal estradiol patch (Estrana TAPE 0.72 mg\\u0026reg;, Hisamitsu Pharmaceutical, Tokyo, Japan), started on the third day of the menstrual cycle or three days after withdrawal bleeding until the day of pregnancy test. 90 mg vaginal progesterone gel (OneCrinone\\u0026reg;; Merck BioPharma, Tokyo) and 30mg/day of dydrogesterone tablets were administered, starting from day 13 of menstrual cycle. The thawed BT was performed five days after commencement of progesterone treatment [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Transdermal estradiol patch, dydrogesterone tablets, and vaginal progesterone gel were continued until the day of pregnancy test, and after positive pregnancy test was verified, the patient maintained the same treatment protocol until 9\\u0026ndash;10 weeks of gestation.\\u003c/p\\u003e\\u003cp\\u003eWe prepared the embryo transfer dish the day before thawed BT, in which 1.0 ml of the embryo transfer medium was taken to the transfer dish and equilibrated for more than 18 hours in an incubator set at 37\\u0026deg;C supplied with a mixed gas of 6% CO2, 5% O2 and 89% N2. EmbryoGlue\\u0026reg; (Vitrolife, Sweden) was used for the HETM group while continuous single culture medium (CSC-C, Fuji Film, Japan) was used for the control group without additional hyaluronan.\\u003c/p\\u003e\\u003cp\\u003eOn the day of thawed BT, the cryopreserved euploid blastocysts were warmed and checked for the viability. Some of those embryos verified alive had a small hole created in the zonae pellucida by LAH (Saturn 5\\u003csup\\u003eTM \\u003c/sup\\u003eACTIVE; Cooper Surgical) before recovery culture for several hours. Blastocysts were assessed again for the morphological grades using Gardner\\u0026rsquo;s Classification [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e] just prior to transfer. Blastocysts which remained a 4BB or better after the thawing process were marked as morphologically good blastocysts, while those blastocysts with C grade in either trophectoderm or inner cell mass (ICM) were marked as morphologically poor blastocysts.\\u003c/p\\u003e\\u003cp\\u003eThe euploid blastocyst selected for transfer were placed in the transfer dish, prepared with equilibrated HETM or control medium, and incubated for a maximum of one-half hours. The euploid blastocyst were replaced into the patient\\u0026rsquo;s uterus transcervically using a soft catheter (Kitazato ET catheter, Kitazato Supply, Shizuoka, Japan) under transvaginal ultrasonography. A single selected blastocyst was replaced into the patient\\u0026rsquo;s uterus transcervically using a soft catheter (Kitazato ET catheter, Kitazato Supply, Shizuoka, Japan) while being monitored under transvaginal ultrasonography.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eClinical outcomes\\u003c/h2\\u003e\\u003cp\\u003eThe pregnancy test was performed 9 days after the thawed BT by measuring serum human chorionic gonadotropin (hCG), where the positive pregnancy test was defined as more than 10 IU/ml of serum hCG. A clinical pregnancy was recognized when the development of a gestational sac (GS) was verified by transvaginal ultrasound imaging 16\\u0026ndash;21 days after the thawed ET. A miscarriage was defined when the fetal heart movement was unable to be confirmed by transvaginal ultrasound imaging in 9 weeks after gestation. The clinical pregnancy rate (CPR) was calculated from the number of cycles with confirmed GS and the total cycle number of thawed BT. The miscarriage rate (MR) was calculated from the number of the miscarriage cycles and the total number of the clinical pregnancies.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e\\u003cp\\u003eThe data acquired in the clinic was statistically analyzed by Fisher\\u0026rsquo;s exact test or non-parametric test. A multivariable logistic regression model was constructed in order to find independent risk factors and complications associated with the pregnancy outcomes after thawed BT using morphologically poor euploid blastocyst, while controlling for confounders. Odds ratios (OR) and their 95% confidence interval (CI) were computed. A p-value of \\u0026lt;\\u0026thinsp;.05 was considered statistically significant. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e].\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003ePatient\\u0026rsquo;s characteristics shown in the Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. The median age of the control and HETM groups were both 39.0 with no significant difference (p\\u0026thinsp;=\\u0026thinsp;.72). The proportions of patients in advanced maternal age (\\u0026ge;\\u0026thinsp;40 years old) in the control and HETM use groups were 40.4% and 42.1%, respectively, without significant difference (p\\u0026thinsp;=\\u0026thinsp;1). The proportion of RIF patients in the HETM group was 50.0%, which was significantly higher than the control group (25.5%, p\\u0026thinsp;=\\u0026thinsp;.025), while the proportions of PRL patients were comparable (p\\u0026thinsp;=\\u0026thinsp;.447). The median number of previous ET attempts in the control and HETM groups were 1 and 2, respectively, but the difference was not statistically significant (p=. 22). Conventional insemination method was used less frequently than ICSI (31.8% and 68.2%, respectively, p\\u0026thinsp;=\\u0026thinsp;.065), but we considered the difference was not significant enough to influence the overall outcomes. In the endometrial preparation before embryo transfer, the NOC protocol was selected for approximately half of the participants (56.5%), while the HRC protocol was selected for another half (43.5%) of the participants (p\\u0026thinsp;=\\u0026thinsp;.66). The proportions of patients underwent LAH after thawing was also similar in these two groups (29.8% in the control and 28.9% in the HETM groups, respectively, p\\u0026thinsp;=\\u0026thinsp;1.0)\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eThe backgrounds of the morphologically poor euploid blastocyst transfer with or without HETM.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"4\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eControl\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eHETM\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eP value\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eNumber of thawed BT\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e47\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e38\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAge, years* [IQR**]\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e39.0 [37.0\\u0026ndash;40.0]\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e39.0 [34.0-40.8]\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.72\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eProportion of AMA\\u003csup\\u003e#\\u003c/sup\\u003e (\\u0026ge;\\u0026thinsp;40), n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e19 (40.4)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e16 (42.1)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e1.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eProportion of RIF\\u003csup\\u003e##\\u003c/sup\\u003e, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e12 (25.5)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e19 (50.0)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.025\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eProportion of PRL\\u003csup\\u003e###\\u003c/sup\\u003e, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e0 (0)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1 (2.6)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.447\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePrevious ET attempts, n* [range, times]\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1 [0\\u0026ndash;3]\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e2 [0\\u0026ndash;5]\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.22\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eMode of insemination (IVF/ICSI), n\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e19/28\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e8/30\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.065\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eEndometrial preparation (NOC/HRC) \\u003csup\\u003eǂ\\u003c/sup\\u003e, n\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e28/19\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e20/18\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.66\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eProportion of LAH \\u003csup\\u003eǂǂ\\u003c/sup\\u003e, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e14 (29.8)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e11 (28.9)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e1.0\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003ctfoot\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"4\\\"\\u003e*median, **IQR; interquartile range\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"4\\\"\\u003e\\u003csup\\u003e#\\u003c/sup\\u003eAMA; advanced maternal age, \\u003csup\\u003e##\\u003c/sup\\u003e RIF; repeated implantation failure, \\u003csup\\u003e###\\u003c/sup\\u003e RPL; recurrent pregnancy loss\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"4\\\"\\u003e\\u003csup\\u003eǂ\\u003c/sup\\u003e NOC/HRC; natural ovulatory cycle/hormone replacement cycle, \\u003csup\\u003eǂǂ\\u003c/sup\\u003e LAH; laser assisted hatching\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tfoot\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe clinical pregnancy rate (CPR) from transfer of a morphologically poor euploid blastocyst was 32.9% (28/85), and it was significantly lower than the CPR from transfer of a morphologically good blastocysts reported by Sato et al. [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. Among the thawed-BT with morphologically poor blastocysts, we found significant improvement in the CPR of HETM group compared to the control group (47.4% [18/38] and 21.3% [10/47], P\\u0026thinsp;=\\u0026thinsp;.019), while we did not see any differences in CPRs by the use of LAH (LAH+; 36.0% [9/25] and LAH-; 31.6% [19/60], respectively, p\\u0026thinsp;=\\u0026thinsp;0.89). (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eThese results led us to identify the predictive factors for achieving a higher chance of pregnancy from the embryo transfer with morphologically poor euploid blastocyst, from a comparison between the group of 28 pregnant patients against the group of 57 non-pregnant patients (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). The average age in the pregnant group was slightly younger than the non-pregnant group (37.5 and 39.0 years, respectively, p\\u0026thinsp;=\\u0026thinsp;.242). There were no significant differences found in either the proportions of advanced maternal age, RIF, PRL, the number of previous ET attempts, or the insemination method. Comparing the endometrial preparation protocols used before thawed BT, the NOC protocol was used more frequently than the HRC protocol (56.4% and 43.6%, respectively). The NOC protocol was used in 67.9% of the pregnant group, which was higher than the non-pregnant group (50.9%, p\\u0026thinsp;=\\u0026thinsp;.76). The proportion of HRC protocol in the pregnant and non-pregnant groups were 32.1% and 49.1%, respectively, and the difference was not significant (p\\u0026thinsp;=\\u0026thinsp;.12). The proportion of embryos underwent LAH method were also comparable between the pregnant and non-pregnant groups (32.15% and 28.1%, respectively, p\\u0026thinsp;=\\u0026thinsp;.801). Interestingly, the proportion of embryos prepared in the HETM medium was 64.3% in the pregnant group, which was significantly higher than the non-pregnant group (35.1%, p\\u0026thinsp;=\\u0026thinsp;.019).\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003ePredictive factors for achieving pregnancy for the morphologically poor euploid blastocyst\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"6\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003ePregnant\\u003c/p\\u003e\\u003cp\\u003egroup\\u003c/p\\u003e\\u003cp\\u003en\\u0026thinsp;=\\u0026thinsp;28\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eNon-pregnant group\\u003c/p\\u003e\\u003cp\\u003en\\u0026thinsp;=\\u0026thinsp;57\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eP-value\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eUnivariate analysis\\u003c/p\\u003e\\u003cp\\u003eOR (95%CI)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eMultivariate analysis\\u003c/p\\u003e\\u003cp\\u003eOR (95%CI)\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eAge, years* [IQR**]\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e37.5 [34.0\\u0026ndash;40.0]\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e39.0 [37.0\\u0026ndash;40.0]\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.242\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0.94 (0.83\\u0026ndash;1.07)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0.96 (0.77\\u0026ndash;1.20)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eProportion of AMA\\u003csup\\u003e#\\u003c/sup\\u003e (\\u0026ge;\\u0026thinsp;40), n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e10 (35.7)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e25 (43.9)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.494\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0.71 (0.28\\u0026ndash;1.81)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0.94 (0.19\\u0026ndash;4.60)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eProportion of RIF\\u003csup\\u003e##\\u003c/sup\\u003e, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e8 (28.6)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e23 (40.4)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.343\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0.59 (0.22\\u0026ndash;1.57)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e1.02 (0.200\\u0026ndash;5.14)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eProportion of PRL\\u003csup\\u003e###\\u003c/sup\\u003e, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e7 (25.0)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e8 (14.0)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.237\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e2.04 (0.66\\u0026ndash;6.34)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e2.53 (0.672\\u0026ndash;9.53)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePrevious ET attempts, n [range, times]\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e0.5 [0.0\\u0026ndash;4.0]\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e2.0 [0.0\\u0026ndash;4.0]\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.087\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0.85 (0.71\\u0026ndash;1.03)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0.83 (0.620\\u0026ndash;1.10)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eMode of insemination (IVF/ICSI), n\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e8/20\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e19/38\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.805\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e1.25 (0.47\\u0026ndash;3.36)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0.87 (0.27\\u0026ndash;2.86)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eEndometrial preparation (NOC\\u0026dagger;/HRC\\u0026dagger;\\u0026dagger;), n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e19/38 (32.1/67.9)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e29/28 (50.9/49.1)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.167\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0.49 (0.19\\u0026ndash;1.27)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e0.47 (0.15\\u0026ndash;1.43)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eProportion of LAH\\u003csup\\u003eǂ\\u003c/sup\\u003e, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e9 (32.1)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e16 (28.1)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.801\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e1.21 (0.46\\u0026ndash;3.24)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e1.59 (0.50\\u0026ndash;5.08)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eHETM\\u003csup\\u003eǂǂ\\u003c/sup\\u003e, n (%)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e18 (64.3)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e20 (35.1)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.019\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e3.33 (1.29\\u0026ndash;8.57)\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003e5.08 (1.62-16.0)\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003ctfoot\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"6\\\"\\u003e*mean, **IQR; interquartile range\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"6\\\"\\u003e\\u003csup\\u003e#\\u003c/sup\\u003e AMA; advanced maternal age, \\u003csup\\u003e##\\u003c/sup\\u003e RIF; repeated implantation failures, \\u003csup\\u003e###\\u003c/sup\\u003e RPL; recurrent pregnancy losses\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"6\\\"\\u003e\\u0026dagger;NOC; natural ovulatory cycle, \\u0026dagger;\\u0026dagger; HRC; hormone replacement cycle,\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd colspan=\\\"6\\\"\\u003e\\u003csup\\u003eǂ\\u003c/sup\\u003eLAH; laser assisted hatching, \\u003csup\\u003eǂǂ\\u003c/sup\\u003eHETM; hyaluronan-enriched transfer medium\\u0026dagger;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tfoot\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe result from univariate analysis indicated that the use of HETM before embryo transfer increased the chance of positive pregnancy outcomes (OR\\u0026thinsp;=\\u0026thinsp;3.33, 95% CI\\u0026thinsp;=\\u0026thinsp;1.29\\u0026ndash;8.57). The multiple logistic regression analysis found that the use of HETM is a predictive factor of positive pregnancy outcomes (OR\\u0026thinsp;=\\u0026thinsp;5.08, 95% CI\\u0026thinsp;=\\u0026thinsp;1.62-16.0).\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe most common way of practice in ART labs to select embryos for blastocyst transfer is based on the morphological evaluation by Gardner\\u0026rsquo;s grading method [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. Timelapse image acquisition systems opened the way to propose alternative methods to select blastocysts for transfer using annotation parameters such as time to reach blastocyst stage, blastocyst expansion speed, ICM and TE grades [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. In addition to embryo selection methods based on morphology, the increasing use of PGT-A allows clinicians to select the blastocysts with higher probability of implantation based on the chromosomal profiles of embryo, or euploidy [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. The potential benefits of combining embryo morphology and PGT-A have been shown in recent publications, that morphological grades could still differentiate the chance of clinical and ongoing pregnancy among euploid embryos [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Our data supports these findings: the clinical pregnancy rate from the transfer of morphologically poor euploid blastocysts was 32.9% (28/85), while the clinical pregnancy rate of morphological good blastocyst was 62.2% (729/1,172, data not shown in the table).\\u003c/p\\u003e\\u003cp\\u003eOur result also demonstrated that HETM group in thawed-BT of morphologically poor euploid blastocysts resulted in much higher success rates than the control group in the same setting. Patient demographics including median age, the proportion of advanced maternal age, and the proportion of PRL, did not correlate with then difference found between these two groups, suggesting that the use of HETM by itself could improve the outcome for morphological poor blastocysts. The proportion of ICSI cycles was slightly higher in the HETM group but the difference was not significant enough. Our recent report found potentially profound impacts of endometrial preparation method to the pregnancy success rate of euploid blastocyst transfer, showing the HRC protocol being a negative predictive factor for clinical pregnancy [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. On the contrary, the proportions of NOC and HRC protocols were quite similar between the control and HETM groups in this study, and there was no bias found regarding the endometrial preparation protocols before embryo transfer. Instead, this study showed that the use of HETM in the preparation of blastocyst before transfer improved the clinical pregnancy rate of morphologically poor euploid blastocyst (47.4% vs. 21.3%, p\\u0026thinsp;=\\u0026thinsp;.0194), indicating that hyaluronan supplementation could help morphologically poor euploid blastocysts implant.\\u003c/p\\u003e\\u003cp\\u003eHyaluronan is one of the essential molecules found in key reproductive events including ovulation, fertilization, embryogenesis, implantation and trophoblast invasion [\\u003cspan additionalcitationids=\\\"CR23\\\" citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. Hyaluronan is synthesized as a liner polymer structure of glycosaminoglycan which consists of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine, and it was formed in polymer of various molecular sizes up to 10\\u003csup\\u003e7\\u003c/sup\\u003e Da. It is produced predominantly by three isoforms of hyaluronic acid synthase (HAS1-3), each generating polymers of different lengths. Turnover of hyaluronan molecules in physiological environments is relatively short, either by oxidation or enzymatic degradation by a family of hyaluronidases (HYALs) [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. Among three main isoforms of HYALs, hyaluronidase 2 (HYAL2) is expressed on the cell surface with a glycosylphosphatidylinositol (GPI)-anchor attached to the extracellular side of the plasma membrane [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Biological functions of hyaluronan polymers in extracellular matrix are modulated through interaction with a variety of hyaluronan-binding proteins, including the most notable hyaluronan-receptor, CD44 [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. HYAL-2 is involved in the processing of long chain hyaluronan in the extracellular space through the interaction with CD44, and the cleavage of hyaluronan via HYAL-2/CD44 complex is essential to the subsequent receptor-mediated internalization of hyaluronan into the cytoplasm [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. Kong et al. recently demonstrated that successful transition of uterine endometrium for establishment of pregnancy requires selective elimination of pro-inflammatory senescent decidual cells by uterine natural killer cells [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. They also reported that morphologically poor human blastocysts derived from IVF/ICSI express lower levels of HYAL2 compared to morphologically good blastocysts (Gardner\\u0026rsquo;s classification; \\u0026ge;BB, p\\u0026thinsp;=\\u0026thinsp;.003). Moreover, the successfully implanted blastocysts had higher level of HYAL2 expression compared to the blastocysts failed to implant after transfer (p\\u0026thinsp;=\\u0026thinsp;.042). This suggests that the morphologically poor euploid blastocysts in our study also have lower levels of HYAL2 on the cell structure, which could limit the interaction with CD44 to process and internalize extracellular hyaluronan [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. Although the information of average molecular weight of hyaluronan in EmbryoGlue\\u0026reg; is not available, the enriched-hyaluronan in this transfer medium might help facilitate the CD44-mediated internalization process to improve of the implantation of morphologically poor euploid blastocysts. An increase of high molecule weight hyaluronan would also increase the density of extracellular matrix to a level closer to uterine endometrium, which could give rise to an immunotolerant reaction via uterine natural-killer cell [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. These series of factors could confer additive effects to help the implantation of morphologically poor blastocysts.\\u003c/p\\u003e\\u003cp\\u003eAs has been demonstrated by numerous reports and publications, morphologically poor blastocysts often resulted in lower implantation rates, even when only the euploid embryos are selected for transfer. Our study strongly suggested that HETM used at the time of blastocyst transfer can significantly improve the likelihood of morphologically poor euploid blastocysts to achieve clinical pregnancy. This provides a relatively simple yet powerful tool for clinicians and embryologists when they find the morphology of blastocysts has worsened after freeze-and-thaw. Our own experience as well as previously published data strongly suggested that the concentration of hyaluronan, and possibly the chain length of hyaluronan polymers, could be essential factors to consider in selecting embryo transfer medium most suitable to improve the chance of clinical pregnancy. It led us to use EmbryoGlue\\u0026reg; as HETM in our study as the hyaluronan concentration data is disclosed by the manufacturer (0.5mg/ml). The data shown in this study supports our previous hypothesis, that HETM is especially helpful to improve the success rates of those cycles associated with less favorable factors, either in patient characteristics or the embryos to be transferred.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors would like to thank Kathryn Gebhardt for English editing for this paper.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003cstrong\\u003eConflict of interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eKoji Nakagawa, Takashi Horikawa, Yuji Orita, Emi Yamashiro, Hideaki Watanabe, Asako Shirai, Hisayo Kataoka, Keiji Kuroda, Satoru Takamizawa, Rikikazu Sugiyama declare that they have no conflicts of interest. Souichi Ogata in an employee of Vitrolife K.K.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was conducted with the own funding of author\\u0026rsquo;s group without any external source.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003cstrong\\u003eAuthors contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNakagawa K is a principal investigator. Horikawa T, Kataoka H, Kuroda K, and Takamizawa, contributed to collect clinical data, and Yamashiro E, Watanabe E, Watanabe H, and Shirai A contributed to deal with ART procedure and handling embryos. Orita Y performed statistically data analysis. Ogata S is the supervisor of this study. Sugiyama R is organized this study. All of them agree with the content of this manuscript. Everyone in the author list reviewed the final manuscript before the submission.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eHuman Rights Statements and informed consent\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards, and it was reviewed and approved by the institutional review board of Sugiyama Clinic. All patients have received and signed on an informed written consent form before entering the study, and they were also informed for an option to abort from the study at any time during the treatment.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eSchoolcraft WB, Lane M, Stevens, Gardner DK. Increased hyaluronan concentration in the embryo transfer medium results in significant increase in human embryo implantation rate. Fertil Steril. 2002; 76 suppl 3: S5\\u003c/li\\u003e\\n\\u003cli\\u003eFriedler S, Schachter M, Strassburger D, Esther K, Ron-El R, Raziel A. A randomized clinical trial comparing recombinant hyaluronan/recombinant albumin versus human tubal fluid for cleavage stage embryo transfer in patients with multiple IVF-embryo transfer failure. Human Reprod. 2007; 22: 2444\\u0026ndash;8.\\u003c/li\\u003e\\n\\u003cli\\u003eKorosec S, Virant-Klun I, Tomazevic T, Zech NH, Meden-Vrtovec H. Single fresh and frozen\\u0026ndash;thawed blastocyst transfer using hyaluronan-rich transfer medium. Reprod BioMed Online. 2007; 15: 701-7.\\u003c/li\\u003e\\n\\u003cli\\u003eUrman B, Yakin K, Ata B, Isiklar A, Balaban B. Effect of hyaluronan-enriched transfer medium on implantation and pregnancy rates after day 3 and day 5 embryo transfers: a prospective randomized study. Fertil Steril. 2008; 90: 604\\u0026ndash;12.\\u003c/li\\u003e\\n\\u003cli\\u003eNakagawa K, Takahashi C, Nishi Y, Juen H, Sugiyama R, Kuribayahsi Y, Sugiyama R. Hyaluronan-enriched transfer medium improves outcome in patients with multiple embryo transfer failures. J Assist Reprod Genet. 2012: 29: 679-85. \\u003c/li\\u003e\\n\\u003cli\\u003eHeymann D, vidal L, Or Y, Shoham Z. Hyaluronic acid in embryo transfer media for assisted reproductive technologies. Cochrane Database of Systematic Reviews 2020, CD007421. DOI: 10.1002/14651858.\\u003c/li\\u003e\\n\\u003cli\\u003ePenzias A, Bendikson K, Butts S, Coutifaris C, Falcone T, Fossum G, Gitlin S, Gracia Hansen K, La Barbera A, Mersereau J, Odem R, Paulson R, Pfeifer S, Pisarska M, Rebar R, Reindollar R, Rosen M, Sandlow J, Vernon M, Widra E. The use of preimplantation genetic testing for aneuploidy (PGT-A): a committee opinion. Fertil Steril. 2018; 109:429\\u0026ndash;36. \\u003c/li\\u003e\\n\\u003cli\\u003eHorikawa T, Nakagawa K, Tonouchi E, Kuroda K, Watanabe H, Takamizawa S, Sugiyama R. Trophectoderm grade and the day of TE biopsy significantly corelate with pregnancy rate in euploid blastocyst transfer. Fertil Steril 2021; 116: e247 \\u003c/li\\u003e\\n\\u003cli\\u003ePGDIS. PGDIS Position Statement on Chromosome Mosaicism and Preimplantation Aneuploidy Testing at the Blastocyst Stage. 2016;1\\u0026ndash;2. \\u003c/li\\u003e\\n\\u003cli\\u003eGardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril. 2000; 73: 1155-8. \\u003c/li\\u003e\\n\\u003cli\\u003eSugiyama R, Nakagawa K, Nishi Y, Ojiro Y, Juen H, Sugiyama R, Kuribayashi Y. Using a mild stimulation protocol combined with clomiphene citrate and recombinant follicle-stimulating hormone to determine the optimal number of oocytes needed to achieve pregnancy and reduce the concerns of patients. Reprod Med Biol. 2013; 12:105\\u0026ndash;10.\\u003c/li\\u003e\\n\\u003cli\\u003eNakagawa K, Oba M, Ehara K, Ishigaki N, Ino N, Itakura A, Ysutsumi R, Nakao K, Ojiro Y, Sugiyama R. Clinical outcomes of assisted reproductive technology treatment by using a self-injection of recombinant human chorionic gonadotropin as the final maturation trigger. Reprod Med Biol. 2018; 17: 203\\u0026ndash;8.\\u003c/li\\u003e\\n\\u003cli\\u003eHiraoka K, Kitamura S. Clinical efficiency of Piezo-ICSI using micropipettes with a wall thickness of 0.625 \\u0026mu;m. J Assist Reprod Genet. 2015; 32: 1827\\u0026ndash;33.\\u003c/li\\u003e\\n\\u003cli\\u003eNakagawa K, Shirai A, Nishi Y, Sugiyama R, Kuribayashi Y, Sugiyama R, Inoue M. A study of the effect of an extremely low oxygen concentration on the development of human embryos in assisted reproductive technology. Reprod Med Biol. 2010; 9: 163-8.\\u003c/li\\u003e\\n\\u003cli\\u003eYang D, Feng D, Gao Y, Sagnelli M, Wang X, Li D. An effective method for trophectoderm biopsy using mechanical blunt dissection: a step-by-step demonstration. Fertil Steril. 2020; 114: 438\\u0026ndash;439.\\u003c/li\\u003e\\n\\u003cli\\u003eSugiyama R, Nakagawa K, Shirai A, Sugiyama R, Nishi Y, Kuribayashi Y, Inoue M. Clinical outcomes resulting from the transfer of vitrified human embryos using a new device for cryopreservation (plastic glade). J Assist Reprod Genet. 2010; 27: 161-7\\u003c/li\\u003e\\n\\u003cli\\u003ePractice Committee of the American Society for Reproductive Medicine. A review of best practices of rapid-cooling vitrification for oocytes and embryos: a committee opinion. Fertil Steril. 2021; 115: 305\\u0026ndash;10.\\u003c/li\\u003e\\n\\u003cli\\u003eKanda Y. Investigation of the freely available easy-to-use software \\u0026ldquo;EZR\\u0026rdquo; for medical statistics. Bone Marrow Transplantation. 2013; 48: 452\\u0026ndash;8.\\u003c/li\\u003e\\n\\u003cli\\u003eSato T, Sugiura-Ogasawara M, Ozawa F, Yamamoto T, Kato T, Kurahashi H, Kuroda T, Aoyama N, Kato K, Kobayashi R, Fukuda A, Utsunomiya T, Kuwahara A, Saito H, Irahara M. Preimplantation genetic testing for aneuploidy: a comparison of live birth rates in patients with recurrent pregnancy loss due to embryonic aneuploidy or recurrent implantation failure. Hum Reprod. 2019; 34: 2340-8. \\u003c/li\\u003e\\n\\u003cli\\u003eIrani M, Reichman D, Robles A, Melnick A, Davis O, Zaninovic N, Xu K, Rosenwaks Z. Morphologic grading of euploid blastocysts influences implantation and ongoing pregnancy rates. Fertil Steril. 2017; 107: 664-70.\\u003c/li\\u003e\\n\\u003cli\\u003eGonzalez XV, Odia R, Naja R, Serhal P, Saab W, Seshadri S, Ben-Nagi J. Euploid blastocysts implant irrespective of their morphology after NGS-(PGT-A) testing in advanced maternal age patients. J Assist Reprod Gent. 2019; 36: 1623-9. \\u003c/li\\u003e\\n\\u003cli\\u003eFouladi-Nashta AA, Raheem KA, Marei WF, Ghafari F, Hartshorne GM. Regulation and roles of the hyaluronan system in mammalian reproduction. Reproduction. 2017; 153: R43-R58, doi:10.1530/REP-16-0240.\\u003c/li\\u003e\\n\\u003cli\\u003eRuane PT, Buck CJ, Babbington PA, Aboussahoud W, Berneau SC, Wwetwood M, Kimber SJ, Aplin JD, Brison DR.\\u003cem\\u003e \\u003c/em\\u003eThe effects of hyaluronate-containing medium on human embryo attachment to endometrial epithelial cells in vitro. Hum Reprod Open.\\u003cstrong\\u003e \\u003c/strong\\u003e2020; hoz033, doi:10.1093/hropen/hoz03.\\u003c/li\\u003e\\n\\u003cli\\u003eTakahashi H, Takizawa T, Matsubara S, ohkuchi A, Kuwata T, Usui R, Matsumoto H, Sato Y, Fujiwara H, Okamoto A, Suzuki M, Takizawa T. Extravillous trophoblast cell invasion is promoted by the CD44- hyaluronic acid interaction. Placenta. 2014;\\u003cem\\u003e \\u003c/em\\u003e35:163-170.\\u003c/li\\u003e\\n\\u003cli\\u003eDicker KT, Gurski LA, Pradhan-Bhatt S, Witt RL, Farach-Carson MC, Jia X. Hyluronan: A Simple Polysaccharide with Diverse Biological Functions. Acta Biomater. 2014; 10: 1558-70.\\u003c/li\\u003e\\n\\u003cli\\u003eRai SK, Duh FM, Vigdorovich V, Danilkovitch-Miagkova A, Lerman MI, Miller AD. Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation. Proc. Natl. Acad. Sci. U S A. 2001; 98:4443\\u0026ndash;4448.\\u003c/li\\u003e\\n\\u003cli\\u003eYang B, Yang BL, Savani RC, Turley EA. Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein. EMBO J. 1994, 13:286\\u0026ndash;296.\\u003c/li\\u003e\\n\\u003cli\\u003eHarada H. and Takahashi M. CD44-dependent intracellular and extracellular catabolism of hyaluronic acid by hyaluronidase-1 and -2. J. Biol. Chem. 2007, 282(8): 5597-5607.\\u003c/li\\u003e\\n\\u003cli\\u003eKong CS, Ordo\\u0026ntilde;ez AA, Turner S, Tremaine T, Muter J, Lucas ES, Salisbury E, Vassena R, Fouladi-Nashta AA, Tiscornia G, Hartshorne G, Brosens JJ, Brighton PJ. Involvement of uterine natural killer cells in the natural selection of human embryos at implantation. FASEB J. 2021, 35 (4): e21336.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":true,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"archives-of-gynecology-and-obstetrics\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"arch\",\"sideBox\":\"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/arch/default.aspx\",\"title\":\"Archives of Gynecology and Obstetrics\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Euploid blastocyst, Hyaluronan, HETM, morphologically poor blastocyst, pregnancy rate\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-2427879/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-2427879/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003ePurpose:\\u003c/h2\\u003e\\n\\u003cp\\u003eHyaluronan-enriched transfer medium (HETM) could improve the clinical pregnancy rate (CPR) for patients with repeated implantation failures (RIF). In contrast, there have been seldom reports addressing the potentially beneficial effects of HETM for morphologically poor blastocysts (MPBLs). Our study aimed to evaluate whether the use of HETM would improve the CPR for the patients who were transferred with euploid MPBLs.\\u003c/p\\u003e\\n\\u003ch2\\u003eMethods:\\u003c/h2\\u003e\\n\\u003cp\\u003ePatients who underwent single euploid blastocyst transfer between July 2020 and June 2022 were enrolled. We included only those blastocysts confirmed as euploid by PGT-A, and those blastocysts were transferred after thawing. The natural ovulatory cycle or hormone replacement cycle (HRC) protocol were used for endometrial preparation for thawed blastocyst transfer (thawed-BT). A total of 1,168 thawed-BT cycles were performed in the study period, including 954 cycles of morphologically good blastocysts (≥ 4BB in Gardner’s classification), and 85 cycles of MPBLs, of which 47 were transferred using HETM in thawed-BT (the HETM group), and the remaining 38 were transferred with the medium without hyaluronan (the control group). We compared the CPR between these two groups.\\u003c/p\\u003e\\n\\u003ch2\\u003eResults:\\u003c/h2\\u003e\\n\\u003cp\\u003eThe characteristics of patients were similar between the HETM and control groups. The CPR in the HETM group was significantly higher than the control group (47.4% and 21.5%, respectively, p = 0.019). The multiple logistic regression analysis found that the use of HETM was a predictive factor of positive pregnancy outcomes (OR = 5.08, 95% CI = 1.62-16.0, p = 0.019).\\u003c/p\\u003e\\n\\u003ch2\\u003eConclusion:\\u003c/h2\\u003e\\n\\u003cp\\u003eOur data suggests that HETM used in the euploid blastocyst transfer can improve the clinical pregnancy rates of morphologically poor blastocysts.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Hyaluronan-enriched transfer medium (HETM) can improve the implantation rate in morphologically poor euploid blastocyst transfer\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2023-01-13 21:02:11\",\"doi\":\"10.21203/rs.3.rs-2427879/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2023-01-19T14:51:14+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2023-01-11T14:15:15+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"Archives of Gynecology and Obstetrics\",\"date\":\"2023-01-11T13:02:48+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2022-12-30T09:50:58+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Archives of Gynecology and Obstetrics\",\"date\":\"2022-12-30T04:17:01+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"archives-of-gynecology-and-obstetrics\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"arch\",\"sideBox\":\"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/arch/default.aspx\",\"title\":\"Archives of Gynecology and Obstetrics\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"2ed086ef-f3e5-4b0e-a943-d6f4fad53e71\",\"owner\":[],\"postedDate\":\"January 13th, 2023\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2023-10-16T21:35:55+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-2427879\",\"link\":\"https://doi.org/10.1007/s00404-023-07083-9\",\"journal\":{\"identity\":\"archives-of-gynecology-and-obstetrics\",\"isVorOnly\":false,\"title\":\"Archives of Gynecology and Obstetrics\"},\"publishedOn\":\"2023-05-31 21:03:22\",\"publishedOnDateReadable\":\"May 31st, 2023\"},\"versionCreatedAt\":\"2023-01-13 21:02:11\",\"video\":\"\",\"vorDoi\":\"10.1007/s00404-023-07083-9\",\"vorDoiUrl\":\"https://doi.org/10.1007/s00404-023-07083-9\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-2427879\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-2427879\",\"identity\":\"rs-2427879\",\"version\":[\"v1\"]},\"buildId\":\"-HB7Z8yhvgn0wM9Nzuekk\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}