P–032 Assessment of embryonic developmental outcome of direct unequal cleavage in patients with non-obstructive azoospermia and/or obstructive azoospermia

Study question Does direct unequal cleavage (DC) affect embryonic development after ICSI with testicular sperm (TESE-ICSI) in patients with non-obstructive azoospermia (NOA) and/or obstructive azoospermia (OA)? Summary answer The incidence of DC at the first cleavage (DC1) was extremely high and DC1 negatively affected embryonic development in NOA patients. What is known already It has been reported that the blastocyst development of embryos with direct cleavage (DC) was significantly lower than that without DC, but the clinical pregnancy rate after blastocyst transfer was not different with or without DC. The incidence of DC has been reported to be significantly higher after ICSI with testicular sperm (TESE-ICSI) than ICSI with ejaculated sperm (Ej), but to our knowledge, there are few reports investigating that the embryos with DC after TESE-ICSI affect embryonic development. Study design, size, duration We conducted a retrospective cohort study using time-lapse incubators (Geri, Genea Biomedx, Australia) from September 2018 to November 2020. Of 1033 two-pronuclear (2PN) embryos from TESE-ICSI, 486 and 547 embryos were from OA (35.9±5.5 years) and NOA (33.7±5.2 years), respectively. As an age matched control, we chose 581 embryos from ICSI using Ej (36.5±4.4 years). Participants/materials, setting, methods DC embryos were classified as DC1 (DC at first cleavage), DC2 (DC at second cleavage), and non-DC (without DC). The incidences of DC1 or DC2 and blastocyst development rates were compared among OA, NOA and Ej groups. In TESE-ICSI group, we compared blastocyst development rates with or without DC between good and poor quality embryos on day 3. Good quality embryos were defined as 8 cells with G3 or more by the Veeck’s classification. Main results and the role of chance DC1 incidence was significantly higher in NOA (37.3%) than OA (27.8%) and Ej (22.7%) (P < 0.01), whereas DC2 incidence was not statistically different among three groups; NOA (15.7%), OA (15.0%) and Ej (13.4%). Blastocyst development rates in DC1 were 17.8%, 19.5% and 25.8% for NOA, OA and Ej, respectively, which were significantly lower compared to non-DC in corresponding three groups (65.1%, 67.7%, and 68.5%, respectively, P < 0.01). In TESE-ICSI group, good-quality embryo rate on day 3 was significantly lower in DC1 (34.5%, P < 0.01) than DC2 (60.9%) or non-DC (54.2%). Additionally, blastocyst development rates in DC1 and DC2 were significantly lower than non-DC regardless of embryonic grades on day 3 (35.1%, 51.0%, and 81.6% for good-quality embryos on day 3, 10.1%, 27.0%, and 49.1% for poor-quality embryos on day 3, respectively, P < 0.05). When immotile sperm was used for TESE-ICSI, DC1 incidence was 40.0% (6/15), which did not show statistically differences. When performing single frozen-thawed blastocyst transfers, no pregnancies resulted from either DC1 (n = 13) or DC2 (n = 3) embryos in TESE-ICSI group. Limitations, reasons for caution We had a few data about the pregnancy rates after blastocyst transfers with DC, because embryos with DC were seldom transferred due to those lower priority. Although DC might be influenced by the sperm, we did not analyze the incidence of DC by taking the semen factors into account. Wider implications of the findings: The incidence of DC1 was extremely high and DC1 negatively affected embryonic development in NOA patients. Therefore, it is important to observe embryos using time-lapse incubator in order to recognize embryos with/without pregnancy potential, especially for embryos with DC1 in NOA patients. Trial registration number Not applicable

P–155 Oocyte recovery 39 hours (from 39h to 41h) after administration of follicular maturation trigger does not affect clinical results

Study question What is the clinical outcome of oocytes recovered after 39 hours from ovulation inducing drug administration? Summary answer Oocytes obtained after 39 hours from follicular maturation triggering are equally viable to those obtained at the standard time of 36 hrs. What is known already In the clinical setting of ART, ovum pick-up (OPU) is generally performed around 36 hours after the administration of ovulation inducing drugs (OID). However, there are cases where OPU cannot be performed at this time often due to long operating lists. As the time elapsed between the administration of ovulation inducing drugs and OPU becomes longer, there is a concern about time-related oocyte aging. Nevertheless, there are few reports of clinical results of OPU after 36 hours from OID. Study design, size, duration We conducted a review of 1187 cycles and 1951 patients in which OPU and embryo transfer was performed in 2017–2018. All cycles underwent a ‘freeze-all’ of embryos and the transfer cycle was in the thawed embryo transfer cycle for all cases. Participants/materials, setting, methods The time from the administration of OID to the end of OPU was divided into 36h group and over 39h group and the MII and normal fertilization rate of oocytes obtained from OPU after ovarian stimulation were compared. After confirmation of fertilization, the D3 good-quality embryo and the D5 and 6 good-quality blastocyst rates of embryos that continued to be cultured and the pregnancy and miscarriage rates of cleavage-stage embryos and blastocyst transfers were compared. Main results and the role of chance The MII rate in the 36h and >39h groups was 78.1% vs. 80.0%, and the normal fertilization rate was 77.9% vs. 78.1% (ICSI) and 65.4% vs. 67.6% (Conventional-IVF). The D3 good-quality embryo rate (good-quality embryos are embryos with less than 5% fragmentation in 7–9 cells and compaction with more than 50% adhesion between split spheres) was 21.8% vs. 25.3%, the D5 good-quality blastocyst rate (at least 3BB according to Gardner classification) was 33.6% vs. 40.1%, and the D6 good-quality blastocyst rate was 31.1% vs. 37.5%, all of which were not significantly different. The pregnancy rate for cleavage-stage embryo transfer was 26.6% vs. 6.7%, and the miscarriage rate was 25.3% vs. 42.9%, both of which were not significantly different. The pregnancy rate for blastocyst transfer was 45.4% vs. 50.0%, and the miscarriage rate was 22.2% vs. 20.0%, both of which were not significantly different. (The significance difference test was a χ-square test) Limitations, reasons for caution The study was a retrospective study. Wider implications of the findings: Even if OPU is conducted after 36h of the administration of OID, to the extreme range of 39h–41h, oocyte aging does not seem apparent and pregnancy outcomes are similar to the standard time interval of 36 hours. Trial registration number ‘not applicable’

Recurrent Implantation Failure: The Role of Anatomical Causes

Recurrent implantation failure (RIF) is one of the great challenges of current reproductive medicine. The term refers to the failure of repeated transfers of embryos of good morphological quality. Embryo implantation is a crucial moment in in vitro fertilization (IVF) treatments. A successful pregnancy depends on a synchronized interaction between a good quality embryo and a receptive endometrium. Its failure may be a consequence of embryo quality, anatomical or immunological factors. The anatomic causes constitute an important factor for RIF, although they are usually manageable. Fibroids, polyps and adhesions that develop after a surgical procedure or infection can hamper the embryo - endometrium attachment process. In addition, Mullerian abnormalities and hydrosalpinx can cause a negative impact on implantation rates and should also be taken into account in patients with RIF. In this chapter, we will address the main anatomical causes that may impact the implantation rates of patients undergoing IVF, as well as recommendations on management and its treatment.

miR-320a-3p Levels in Human Granulosa Cells: A Promising Bio-marker of Good Quality Embryo and Clinical Pregnancy after IVF/ICSI.

BackgroundmiRNAs in body fluids are considered potential biomarkers of diseases. This study investigated whether miR-320a-3p and miR-483-5p levels in human granulosa cells from follicular fluids were associated with embryo developmental competence. MethodsWe collected 195 patients’ granulosa cells samples undergoing in vitro fertilization (n =147) or intracytoplasmic sperm injection (n =48) cycles, and gathered information about the outcomes of the treatment. miR-320a-3p and miR-483-5p levels were measured using qRT-PCR.ResultsThe miR-320a-3p levels in human granulosa cells across different patient groups were significantly different in the good quality embryo rates, with lowest levels in the Q4 intervals (P<0.05). The relative expression levels of miR-320a-3p were negatively associated with clinical pregnancy rate (P<0.05) and positively correlated with the patient age (P=0.0033). Moreover, both the basal FSH (P=0.0003) and ovarian stimulation protocol (P=0.006 and P=0.004) significantly and positively affected miR-320a-3p levels. The days of stimulation was negatively correlated with the relative expression of miR-320a-3p (P=0.0466). The relative expression levels of miR-483-5p were significantly positively correlated with AMH (P=0.0047). Neither miR-320a-3p nor miR-483-5p levels in granulosa cells were associated with normal fertilized rate, blastulation rate and abortion rate. ConclusionsThe miR-320a-3p levels in human granulosa cells were negatively correlated with the good quality embryo rate and clinical pregnancy rate and positively correlated with the patient age, indicating that miR-320a-3p can be used as a potential indicator to predict embryo development ability and clinical pregnancy.

Effect of Day 3 and Day 5/6 Embryo Quality on the Reproductive Outcomes in the Single Vitrified Embryo Transfer Cycles

ObjectiveTo explore the live birth rate and neonatal outcome after single vitrified blastocyst transfer versus single vitrified cleavage-stage embryo transfer at different grades of embryo quality.MethodsA retrospective cohort study including 6077 single vitrified-thawed embryo transfer cycles was performed in the time-period from January 2013 to December 2018.ResultsAfter controlling for potential confounding variables, there are 161% increased odds of a live birth after transfer of single good quality embryo at day 5, 152% increased odds of a live birth after transfer of single poor quality embryo at day 5, 60% increased odds of a live birth after transfer of single good quality embryo at day 6 compared with transfer of single good quality embryo at day 3. Results from the generalized estimated equation regression showed significant relationship of unadjusted birth weight with development stage of embryo and embryo quality (good quality embryo on day 5 vs. Good quality embryo on day 3:β=108.55, SE=34.89, P=0.002; good quality embryo on day 6 vs. Good quality embryo on day 3:β=68.80, SE=33.75, P=0.041). However, no significant differences were seen in birth weight between transfer single poor quality embryo on day 5, 6 and transfer single good quality embryo on day 3.ConclusionA significant increase in live birth rate and birth weight after transfer of single good quality embryo on day 5 and day 6 compared with transfer of single good quality embryo on day 3 in the vitrified embryo transfer cycles.

The Effect of Embryo Transfer Process Duration on Implantation Success

OBJECTIVE: To evaluate the effect of the embryo transfer duration of standard and simple embryo transfer method. STUDY DESIGN: This study was a retrospective cohort study conducted at a tertiary ART Centre, between June 2018- September 2018. Day 5 fresh embryo transferred patients aged between 18 - 40, BMI <35 kg/m2 without uterine pathology were enrolled in the study. Patients were divided into two groups. Group-1 consisted of patients who had successful implantation and Group-2 consisted of patients who did not have implantation. Groups were compared according to their embryo transfer durations. Ninety-two patients were enrolled in the study. Also, sub-steps of as; cleaning of the cervical mucus and placing the outer catheter in the cervix, loading the embryo to the catheter, the period between embryo loading and embryo transfer, and following that, time spent for retracting the outer catheter evaluated. RESULTS: Between Group-1 and Group-2, there was no significant difference for the period of cervical cleaning and placing the outer catheter into the cervix (Respectively; 63 sec vs. 76 sec; p=0.18), the period of embryo loading (Respectively; 69sec vs. 71sec; p=0.46), the period between embryo loading and embryo transfer (Respectively; 10 sec vs. 10 sec; p=0.74, retracing the outer catheter (Respectively; 25.5sec vs. 24sec; p=0.42 and the total period of embryo transfer (182sec vs. 182.5 sec; p=0.55). CONCLUSION: The embryo transfer duration is not related to implantation rates. The duration of the embryo transfer process steps is not a distinguishing factor if a good-quality embryo transfer is done.

Comparison of the predictive value of progesterone-related indicators for pregnancy outcomes of women undergoing the short-acting GnRH agonist long protocol: a retrospective study

Background: There are many progesterone (P) elevation-related indicators for predicting pregnancy outcomes, including the serum P, P-to-oestradiol ratio (P/E2), P-to-follicle index (PFI), and P-to-mature oocyte index (PMOI); however, due to inconsistencies in study populations and controlled ovarian hyperstimulation (COH) protocols among studies, these indicators are controversial. Moreover, no researchers have included these four commonly used indicators in one study to compare their predictive efficacies. The objective of this study was to compare the predictive value of P-related indicators for pregnancy outcomes of women undergoing the short-acting GnRH agonist long protocol. Methods: A total of 612 infertile women undergoing IVF/ICSI were recruited for this study. Serum samples were obtained on the morning of HCG injection for serum P and E2 measurements. Transvaginal ultrasound was performed to determine the follicle count (≥ 14 mm in diameter). The number of mature oocytes was observed in the embryo laboratory after oocyte retrieval.Results: In cases of P<2.5 ng/ml, there was no significant difference in the serum P level or P/E2 between the pregnant group and the non-pregnant group. The PFI and PMOI of the pregnant group were significantly lower than those of the non-pregnant group. According to the stratified analysis of the ovarian response, only the PMI and PMOI of the pregnant women in the normal ovarian response group were lower than those of the non-pregnant women. To compare the predictive value of the PFI and PMOI in IVF/ICSI outcomes, the patients were divided into four groups. The good-quality embryo rate and clinical pregnancy rate were highest in Group A (low PFI and low PMOI) and lowest in Group D (high PFI and high PMOI). In the two groups with discordant PFI and PMOI, namely Group B (low PFI and high PMOI) and Group C (high PFI and low PMOI), the good-quality embryo rate and clinical pregnancy rate were not significantly different.Conclusions: The PFI and PMOI had equal value in predicting clinical pregnancy outcomes in the normal ovarian response group undergoing the short-acting GnRH agonist long protocol. Each clinical centre can choose one of the indicators according to their actual situation in clinical practice and establish individual cut-off values for PFI and PMOI based on their own hormonal measurements.

Comparison of the predictive value of progesterone‐related indicators for pregnancy outcomes of women undergoing the short‐acting GnRH agonist long protocol: a retrospective study

Background There are many progesterone (P) elevation-related indicators for predicting pregnancy outcomes, including the serum P, P-to-oestradiol ratio (P/E2), P-to-follicle index (PFI), and P-to-mature oocyte index (PMOI); however, due to inconsistencies in study populations and controlled ovarian hyperstimulation (COH) protocols among studies, these indicators are controversial. Moreover, no researchers have included these four commonly used indicators in one study to compare their predictive efficacies. The objective of this study was to compare the predictive value of P-related indicators for pregnancy outcomes of women undergoing the short-acting GnRH agonist long protocol. Methods A total of 612 infertile women undergoing IVF/ICSI were recruited for this study. Serum samples were obtained on the morning of HCG injection for serum P and E2 measurements. Transvaginal ultrasound was performed to determine the follicle count (≥ 14 mm in diameter). The number of mature oocytes was observed in the embryo laboratory after oocyte retrieval. Results In cases of P < 2.5 ng/ml, there was no significant difference in the serum P level or P/E2 between the pregnant group and the non-pregnant group. The PFI and PMOI of the pregnant group were significantly lower than those of the non-pregnant group. According to the stratified analysis of the ovarian response, only the PMI and PMOI of the pregnant women in the normal ovarian response group were lower than those of the non-pregnant women. To compare the predictive value of the PFI and PMOI in IVF/ICSI outcomes, the patients were divided into four groups. The good-quality embryo rate and clinical pregnancy rate were highest in Group A (low PFI and low PMOI) and lowest in Group D (high PFI and high PMOI). In the two groups with discordant PFI and PMOI, namely Group B (low PFI and high PMOI) and Group C (high PFI and low PMOI), the good-quality embryo rate and clinical pregnancy rate were not significantly different. Conclusions The PFI and PMOI had equal value in predicting clinical pregnancy outcomes in the normal ovarian response group undergoing the short-acting GnRH agonist long protocol. Each clinical centre can choose one of the indicators according to their actual situation in clinical practice and establish individual cut-off values for PFI and PMOI based on their own hormonal measurements.

Transferrin and antioxidants partly prevented mouse oocyte oxidative damage induced by exposure of cumulus-oocyte complexes to endometrioma fluid

Background Exposure of oocytes to the endometrioma fluid has an adverse effect on embryonic quality. To determine whether adding transferrin and antioxidants to culture medium could counteract detrimental effects on mouse cumulus-oocyte complexes (COCs) induced by exposure to endometrioma fluid or not, we conducted an in vitro cross-sectional study using human and mouse COCs. Methods Eighteen women who had their oocytes exposed to endometrioma fluid during oocyte retrieval were enrolled. COCs from superovulated ICR female mice were collected. They were first exposed to human endometrioma fluid and then treated by transferrin and/or antioxidants (cysteamine + cystine). Subsequently, COCs function was assessed by molecular methods. Results This study observed that human COCs inadvertently exposed to endometrioma fluid in the in vitro fertilization (IVF) group led to a lower good quality embryo rate compared to intracytoplasmic sperm injection (ICSI) group. Exposure of mouse COCs to endometrioma fluid accelerated oocyte oxidative damage, evidenced by significantly reduced CCs viability, defective mitochondrial function, decreased GSH content and increased ROS level, associated with the significantly higher pro-portion of abnormal spindles and lower blastocyst formation (p < 0.05, respectively). This damage could be recovered partly by treating COCs with transferrin and antioxidants (cysteamine + cystine). Conclusions Transferrin and antioxidants could reduce the oxidative damage caused by COCs exposure to endometrioma fluid. This finding provides a promising new possibility for intervention in the human oocyte oxidative damage process induced by endometrioma fluid during oocyte pick-up.