Conventional IVF Research Articles

Influence of the sperm DNA fragmentation index on the outcome of rescue ICSI and the clinical value of rescue ICSI.

As a remedy for the failure of in vitro fertilization (IVF), rescue intracytoplasmic sperm injection (R-ICSI) has been widely carried out, but it has failed to significantly improve the fertilization rate and clinical pregnancy rate. Sperm DNA fragmentation index (DFI) was highly correlated with pregnancy outcome of artificial assisted reproduction. This study aims to investigate the effect of the sperm DFI on the outcome of R-ICSI and the clinical value of R-ICSI. This retrospective analysis was conducted among 140 infertile couples receiving R-ICSI in from January 2014 to December 2019. The subjects were assigned into a total fertilization failure (TFF)+low DFI group (R-ICSI after TFF and DFI<30%) (n=63), a TFF+high DFI group (R-ICSI after TFF and DFI≥30%) (n=16), a partial fertilization failure (PFF)+low DFI group (R-ICSI after PFF and DFI<30%) (n=52), a PFF+high DFI group (R-ICSI after PFF and DFI≥30%) (n=9). All transferred embryos were come from R-ICSI. The general clinical data [infertility duration, male age, female age, basal serum level of follicle stimulating hormone (FSH), basal serum level of luteinizing hormone (LH), antral follicle count, endometrial thickness of human chorionic gonadotropin (HCG) day, and eggs] and R-ICSI cycle outcomes (fertilization rate, normal fertilization rate, cleavage rate, good embryo rate, implantation rate, clinical pregnancy rate and live birth rate) were analyzed. In addition, the effect of R-ICSI on the fertilization outcome of conventional IVF total fertilization failure and partial fertilization failure was explored. There was no significant difference in the general clinical data and R-ICSI cycle outcome between the TFF+low DFI group and the TFF+high DFI group (all P>0.05). There was no significant difference in the general clinical data between the PFF+low DFI group and the PFF+high DFI group (all P>0.05). The fertilization rate and normal fertilization rate in the PFF+low DFI group were significantly higher than those in the PFF+high DFI group (85.40% vs 72.41%, 71.90% vs 58.62%, respectively; both P<0.05). However, there was no significant difference in cleavage rate, good embryo rate, implantation rate, clinical pregnancy rate, and live birth rate between the 2 groups (all P>0.05). The R-ICSI cycle of TFF: A total of 79 fresh cycles, 57 fresh transplant cycles, a total of 761 unfertilized oocytes, and 584 M II oocytes were treated with R-ICSI, the fertilization rate was 83.22%, the normal fertilization rate was 75.51%, the cleavage rate was 98.15%, the good embryo rate was 40.74%, the implantation rate was 30.56%, and the clinical pregnancy rate was 43.86%; 29 live births were obtained. The R-ICSI cycle of PFF: A total of 61 fresh cycles, 31 fresh transplant cycles, a total of 721 unfertilized oocytes, and 546 M II oocytes were treated with R-ICSI; the fertilization rate was 83.33%, the normal fertilization rate was 69.78%, the cleavage rate was 97.36%, the good embryo rate was 44.39%, the implantation rate was 25.42%, and the clinical pregnancy rate was 45.16%; 12 live births were obtained. In the case of partial fertilization failure of IVF, the sperm DFI affects the fertilization rate and normal fertilization rate of R-ICSI; whether it is a TFF of IVF or PFF of IVF, ICSI can be used as an effective remedy way.

The Effect of Uterine Adenomyosis on IVF Outcomes: a Systematic Review and Meta-analysis.

To investigate the impact of uterine adenomyosis on in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) clinical outcomes and also to evaluate the impact of gonadotropin-releasing hormone agonist (GnRHa) and surgical treatments. Infertile women with adenomyosis undergoing conventional IVF or ICSI were compared with women without adenomyosis. Fertility outcomes were evaluated in two patients with adenomyosis untreated and treated surgically or medically therapy. The review protocol was registered in PROSPERO (CRD42020214586). We consider the live birth rate (LBR) as the primary outcome. The analysis showed that women with adenomyosis had lower LBR (OR 0.59, 95% CI 0.37-0.92, p = 0.02), clinical pregnancy rate (OR 0.66, 95% CI 0.48-0.90), and ongoing pregnancy rate (OR 0.43, 95% CI 0.21-0.88) compared to those without adenomyosis, and miscarriage rate was higher in women with adenomyosis (OR 2.11, 95% CI 1.33-3.33). Surgical treatment increases natural conception in women with adenomyosis although the paucity of data (only two studies), and conversely, treatment with GnRHa does not increase the IVF outcomes. Women with adenomyosis have decreased IVF clinical outcomes. Pretreatment with the use of long-term GnRHa could not be beneficial, even though only three studies were included in the meta-analysis. The major part of the studies has not divided focal and diffuse adenomyosis, and this represents a relevant source of bias: studies conducted with standardized diagnostic criteria for adenomyosis are still needed to determine if the different clinical presentations of such condition could compromise IVF outcomes.

Randomized prospective study comparing conventional In Vitro Fertilization technique to Intravaginal Culture with the INVOCELL™ device for 3 and 5 days.

The objective was to analyse and compare the formation and quality of the embryos developed using conventional in vitro fertilization (IVF) and IVC techniques with an INVOCell™ device. Two groups were formed, with eight couples in each, one in culture for three days (D3) and another in culture for five days (D5), using intravaginal culture technique with an INVOcell device and a conventional in vitro fertilization technique. Embryo formation in Group D5 showed 46.7% (IVC) and 40% (IVF) of recovered blastocysts. In the group D5, the conventional IVF, better embryo development dynamics was observed, with 66% of expanded blastocysts, against 28% in the IVC. Group D3 showed 75% (IVC) and 53% (IVF) of embryo formation. Embryonic quality in Group D3 demonstrated that IVF embryos had a better synchrony in the number and quality of blastomeres. All embryos recovered in Group D3, in both techniques, did not show fragmentation. The pH of the medium contained in the INVOCell™ device in both Groups D5 and D3 showed no differences. The means were 7.26 and 7.25, respectively. The pH of the medium used in IVF was 7.29 in both groups. Microbiological analyzes of the culture media contained in the INVOCell™ devices used in Group D5 were negative. The results showed that the IVC technique, using the INVOCell™ device, provided a healthy and balanced environment for the development and obtaining of quality embryos with three and five days of culture.

Effectiveness and Cost-effectiveness of Minimal Ovarian Stimulation in-vitro Fertilization versus Conventional Ovarian Stimulation in Poor Responders: Economic Evaluation Alongside a Propensity Score Adjusted Prospective Observational Study.

Information on the pregnancy rate after successive in-vitro fertilization (IVF) cycles and their associated costs is relevant for couples undergoing assisted reproduction treatments (ARTs). This study, therefore, sought to investigate the effectiveness and the cost-effectiveness of two ARTs, the minimal ovarian stimulation IVF (MS-IVF) compared to the conventional ovarian stimulation IVF (C-IVF) from the payer's perspective. A 10-months follow-up prospective observational study was conducted in a sample of couples who sought ARTs in a private clinic in Southern Brazil. Women had to satisfy the Bologna Criteria and be older than 35 years. The effect outcome was pregnancy rate per initiated cycle. Medication costs were based on medical records. Costs and effect differences were estimated using seemingly unrelated regressions adjusted for the propensity score estimated based on women's characteristics. All 84 eligible women who agreed to participate received a total of 92 IVF cycles (MS-IVF, n=27[35 cycles]; C-IVF n=57[57 cycles]. The effect difference between MS-IVF and C-IVF was -5.1% (95%CI, -13.2 to 5.2). Medication costs of MS-IVF were significantly lower than C-IVF by €-1260 (95%CI, -1401 to -1118). The probabilities of MS-IVF being cost-effective compared to C-IVF ranged from 1 to 0.76 for willingness-to-pay of €0 to €15,000 per established pregnancy, respectively. Even though there were no positive effect differences between groups, MS-IVF might be cost-effective compared to C-IVF from the payer's perspective due to its relatively large cost savings compared to C-IVF. However, further investigation is needed to confirm these findings in a larger sample.

Two modified natural in vitro fertilisation (IVF) protocols compared to conventional IVF treatment: Retrospective data from one Danish Fertility Centre

Aim of study: Over the last decade, laboratory procedures in in vitro fertilisation (IVF) have improved. Hyperstimulated ovaries cause an overload of surplus embryos. The present study was designed to evaluate the efficiency of two different modified IVF cycle protocols trying to reduce the load of medication used for IVF: simple IVF (S-IVF), Tamoxifen 40 mg daily from day 2 in the cycle to ovulation induction) and mild IVF (M-IVF), Tamoxifen 40 mg daily and every secondary 150 IU Gonal F until ovulation induction. The study aims to evaluate their efficiency compared with our conventional IVF (C-IVF) using a short antagonist protocol. Methods: A retrospective cohort study including all patients admitted to IVF for unexplained infertility, tubal factor, and male factor. In all stimulated cycles patients aimed at having fresh embryos transferred and surplus good embryos cryopreserved. All patients were recruited in the same period and allocated to the different treatments on their own request. The study was conducted between June 2019 and February 2021. Results: In total the study included 976 IVF cycles. 651 cycles from C-IVF, 145 cycles from S-IVF and 180 cycles from M-IVF. Mean age in the groups were comparable. Mean number of eggs retrieved was 6.1 (C-IVF), 1.2 (S-IVF) and 3.0 (M-IVF). Pregnancy rate per fresh transfer was found to be 29% for C-IVF, 26% in the S-IVF group and for the M-IVF 20%. Conclusion: Modified IVF stimulation protocols may be an important step towards a simpler assisted reproductive technology (ART) approach. More tolerable for women, easier and cheaper for patients and society they maintain acceptable clinical pregnancy rates. Large prospective studies need to be performed in the future.

Live Birth with or without Preimplantation Genetic Testing for Aneuploidy

BackgroundEmbryo selection with preimplantation genetic testing for aneuploidy (PGT-A) may improve pregnancy outcomes after initial embryo transfer. However, it remains uncertain whether PGT-A improves the cumulative live-birth rate as compared with conventional in vitro fertilization (IVF).MethodsIn this multicenter, randomized, controlled trial, we randomly assigned subfertile women with three or more good-quality blastocysts to undergo either PGT-A or conventional IVF; all the women were between 20 and 37 years of age. Three blastocysts were screened by next-generation sequencing in the PGT-A group or were chosen by morphologic criteria in the conventional-IVF group and then were successively transferred one by one. The primary outcome was the cumulative live-birth rate after up to three embryo-transfer procedures within 1 year after randomization. We hypothesized that the use of PGT-A would result in a cumulative live-birth rate that was no more than 7 percentage points higher than the rate after conventional IVF, which would constitute the noninferiority margin for conventional IVF as compared with PGT-A.ResultsA total of 1212 patients underwent randomization, and 606 were assigned to each trial group. Live births occurred in 468 women (77.2%) in the PGT-A group and in 496 (81.8%) in the conventional-IVF group (absolute difference, −4.6 percentage points; 95% confidence interval [CI], −9.2 to −0.0; P<0.001). The cumulative frequency of clinical pregnancy loss was 8.7% and 12.6%, respectively (absolute difference, −3.9 percentage points; 95% CI, −7.5 to −0.2). The incidences of obstetrical or neonatal complications and other adverse events were similar in the two groups.ConclusionsAmong women with three or more good-quality blastocysts, conventional IVF resulted in a cumulative live-birth rate that was noninferior to the rate with PGT-A. (Funded by the National Natural Science Foundation of China and others; ClinicalTrials.gov number, NCT03118141.)

Thyroid function and IVF outcome for different indications of subfertility.

Graphical Studies evaluating pregnancy outcomes after assisted reproductive treatment (ART) in women with high-normal (2.5–4.5 mIU/L) thyroid-stimulating hormone (TSH) levels are conflicting, possibly due to different patient charactistics and subfertility indications. The aim of this study was to examine the hypothesis that high-normal compared to low-normal TSH levels are associated with adverse implications for pregnancy outcomes in conventional in vitro fertilization (IVF)-treated women. Therefore, we analyzed retrospectively the characteristics and pregnancy outcomes of 949 subfertile women with TSH 0.3–4.5 mIU/L, treated with conventional IVF between January 2008 and March 2012. Demographic and baseline characteristics were compared between groups of patients based on TSH quartiles, using one-way Anova, Kruskal–Wallis ANOVA and chi-square test. Women with high-normal quartile TSH were significantly more likely to be primary subfertile (P = 0.01), with a higher prevalence of unexplained subfertility and with 15% fewer live births after IVF compared to lower TSH quartiles (P = 0.02). In secondary subfertile women with high-normal TSH, male factor subfertility prevailed (P = 0.01), with more live births (P = 0.01). When analyzing primary and secondary subfertile women as one group, these differences failed to be observed, showing no differences in cumulative pregnancy outcomes of IVF between TSH quartiles (I: 0.3–1.21 mIU/L; II: 1.22–1.68 mIU/L; III: 1.69–2.31 mIU/L; IV: 2.32–4.5 mIU/L). In conclusion, primary subfertile women predominate in the high-normal TSH quartile, associated with significantly fewer live births in a subgroup of primary unexplained subfertile women (9%; n = 87/949), while in secondary subfertile women, dominated by male factor subfertility, high-normal TSH is associated with more live births.Lay summaryThyroid hormones are required for all cell processes in the body. An underactive thyroid gland, in which insufficient thyroid hormones are produced and thyroid-stimulating hormone (TSH) rises, is associated with a lower chance of pregnancy. It is not yet clear above which TSH level, 4.5 or also 2.5 mIU/L, this lower probability occurs. Therefore, in 949 couples treated with conventional IVF, we examined whether high-normal TSH levels (TSH: 2.5–4.5 mIU/L) compared to low normal TSH levels (0.3–2.5 mIU/L) affect the live birth rate. We found that women who were trying to become pregnant for the first time, especially without any other cause, that is unexplained subfertility, were more likely to have higher TSH levels. These women had a much lower chance of having a baby compared to women with low-normal TSH levels.

VP23.01: Cardiac remodelling in fetuses conceived following natural cycle IVF versus conventional IVF

VP23.01: Cardiac remodelling in fetuses conceived following natural cycle IVF versus conventional IVF

Time-lapse monitoring of fertilized human oocytes focused on the incidence of 0PN embryos in conventional in vitro fertilization cycles

We aimed to investigate why the incidence of embryos derived from oocytes with no pronuclei (0PN) decreases using time-lapse monitoring (TLM) versus fixed-point assessment in conventional IVF cycles. We analyzed 514 embryos monitored with TLM 6–9 h after insemination and 144 embryos monitored using microscopic assessment 18–21 h after insemination. The primary endpoint of this study was the incidence of 0PN-derived embryos in short insemination followed by TLM. The secondary endpoint was the duration of insemination. As exploratory endpoints, we analyzed the blastulation rate and cryo-warmed blastocyst transfer outcome of embryos with early PN fading, whereby PN disappeared within < 20 h following the initiation of insemination. The incidence of 0PN-derived embryo reduced more significantly through TLM than through fixed-point observation. The microscopic assessment time was more significantly delayed in the 0PN-derived embryo than that in the 2PN-derived embryo. The embryo with early PN fading formed good-quality blastocysts, and their pregnancy outcomes were similar to those of other embryos. Most 0PN-derived embryos in the fixed-point assessment might have resulted from missed observation of PN appearance in the early-cleaved embryos. TLM or strict laboratory schedule management may reduce 0PN-derived embryos by reducing missed PN observations.

THE MORPHOLOGICAL DYNAMICS OF BLASTOCYSTS VITRIFICATION IN EQUILIBRIUM SOLUTION: CLINICAL OUTCOMES AND INFLUENCING FACTORS

During blastocyst vitrification, dehydration and permeation of the cryoprotectant cause the blastocyst to shrink and re-expand in the equilibrium solution. But some blastocysts do not re-expand. This study examines the clinical outcomes and influencing factors of embryos without re-expansion. This study is based on data collected on 9083 vitrified/warmed transferred blastocysts between May 2013 to August 2020. Blastocysts that did not re-expand to more than 80% of their former volume in equilibrium solution (15 min) were defined as the NON group (n=470). The clinical results were compared with the blastocysts which re-expanded (RE group; n=8613). The clinical outcomes were evaluated in terms of followings: ① patient characteristics (under 38 years old or 38 years old and over), ②embryo quality (good or poor), ③ the day of blastocyst culture (D5 or D6). In addition, we evaluated which factors influenced those not re-expanded, ④ culture medium (G series or Global) and ⑤ fertilization methods (conventional IVF or ICSI). The pregnancy rates were significantly lower in the NO group (① patient characteristics: under 38 years old, 48.6% vs. 58.0%, over 38 years old and over, 22.5% vs. 38.8%; ② embryo quality: good 43.2% vs. 55.1%, poor 25.2% vs. 35.5%; ③ the day of blastocyst culture: D5 37.6% vs. 51.2%, D6 20.2% vs. 31.4%; p<0.01, respectively). Abortion rates did not differ between the two groups. We compared no re-expansion rates under 5 conditions and obtained results from ① 4.3% vs. 5.9% (under 38 years old, 38 years old and over), ② 3.4% vs. 7.9% (good, poor), ③ 4.8% vs. 7.9% (D5, D6), ④ 7.3% vs. 4.7% (G series, Global), ⑤ 4.9% vs. 5.6% (conventional IVF, ICSI). There were significant differences in maternal age, embryo quality, day of blastocyst culture, and culture medium (p<0.01). During vitrification, the absence of re-expansion has a negative impact on pregnancy rates. This suggests that embryologists should carefully observe the presence or absence of re-expansion in the equilibrium solution during the vitrification, and that it is desirable to preferentially select embryos showing re-expansion in frozen embryo transfer. Further investigation is necessary to search for influencing factors of no re-expansion.

IS IN VIVO BETTER THAN IN VITRO? A COMPARISON OF SIBLING EMBRYO DEVELOPMENT AND PREGNANCY RATES THROUGH INVOCELL AND CONVENTIONAL IVF

To determine whether in vivo embryo development by INVOCELL intravaginal culture system is superior to in vitro by conventional IVF (cIVF) and whether pregnancy outcome is different by using sibling oocytes.

OPTIMAL METHOD AND TIMING TO INSEMINATE OOCYTES GENERATED FROM NUCLEAR TRANSFER

To determine the optimal method and timing to inseminate artificial oocytes produced by nuclear transfer of haploid pseudo-blastomeres (HpBs). Metaphase II (MII) oocytes were harvested from FVB/N mice and treated with 8% ethanol for parthenogenetic activation. Monopronucleated oocytes with extruded polar body (1PN/2PB) were cultured to the 8-cell stage and treated with a reversible microtubule polymerization inhibitor (nocodazole, 10 μM) to synchronize HpBs at the G2/M phase until nuclear transfer. Another cohort of B6D2F1 mouse oocytes were harvested, exposed to 5 μM cytochalasin B, and enucleated to generate donor ooplasts. A single HpB was coated with inactivated Sendai virus and subzonally transferred into the perivitelline space of the donor ooplast. Successfully reconstructed oocytes were either inseminated by conventional IVF immediately (0h-IVF) with C57BL/6 sperm or inseminated by piezo-actuated ICSI 2h (2h-ICSI) after nuclear transfer. Control zygotes were generated by piezo-ICSI of intact oocytes. All oocytes were cultured up to 96h in a time-lapse incubator to obtain and compare morphokinetic data for full preimplantation development. A total of 149 FVB/N oocytes were activated, and 29.5% reached the 8-cell stage 48h later, generating a total of 352 HpBs. Another cohort of 188 B2D2F1 oocytes were enucleated at a survival rate of 98.4%. Nuclear transfer of HpBs yielded a total of 167 oocytes, which were allocated to the 0h-IVF and 2h-ICSI groups. A portion of those allocated to 2h-ICSI spontaneously re-cleaved before ICSI insemination. While no oocyte was degenerated in the 0h-IVF group, the ICSI survival rate of the 2h-ICSI group was comparable to the control-ICSI group (63.0% vs. 67.7%). When comparing fertilization success, the 2h-ICSI group yielded a significantly lower rate (61.5%) than the 0h-IVF group (86.4%, p = 0.009). When comparing development rates to the blastocyst stage, the 2h-ICSI group yielded a rate of 7.7%, while the 0h-IVF group trended to a superior yield of 23.5%, albeit not statistically significant. When blastulation morphokinetic data were compared, all progressing embryos from the 0h-IVF, 2h-ICSI, and control groups showed comparable timing. To date, after transferring 22 morula/blastocysts from the 0h-IVF group, 1 pregnancy was generated, resulting in 3 healthy pups (2 male and 1 female). Our technique can reproduce functional copies of female gametes from a single ovum using a mouse model. While the spindle physiology and cell cycle of these artificial oocytes are not well understood, we determined that conventional IVF is an effective method for insemination, yielding a higher fertilization rate and trending to a higher blastocyst yield.

ASSESSMENT OF SEMEN CHARACTERISTICS IN CONSECUTIVE EJACULATES WITH SHORT ABSTINENCE USING SQA-V SPERM QUALITY ANALYZER

To determine the effect of consecutive ejaculates on seminal parameters after short abstinence using the SQA-V sperm quality analyzer tool. The prospective study was conducted from February 2020 to November 2020 in males undergoing treatment for primary infertility. Fifty-five fresh semen samples from infertile men were obtained by masturbation and analysed. Two semen samples were collected in succession after an abstinence period of 4 days followed by another sample after only 2 hrs. For both ejaculates, semen volume, sperm concentration, total motility & motility groups and detailed kinematic parameters were assessed using SQA-V sperm quality analyzer. The Statistical analysis was carried out by using the software, Statistical Package for Social Science, SPSS 20.0 (SPSS, Chicago, III., USA). The obtained results were analyzed statistically using student’s t-test & P-values <0.05 were considered significant. The second raw ejaculate demonstrated lower semen volume (1.39 ± 0.48 vs 3.0 ± 0.73, p<0.05) and sperm concentration (43.87 ± 14.69 vs 60.54 ± 14.50, p<0.05) than initial sample. While a significant increase in total motility and progressive motility in the consecutive sample than the initial sample (61.6 ± 8.21 vs 54.70 ± 8.38, p<0.05 and 49.83 ± 8.34 vs 43.45 ± 8.45, p<0.05). The findings of the present study indicate that pooling the spermatozoa from second ejaculate may allow for a simple method to select high quality spermatozoa and also could be an advantage for intrauterine insemination (where a large number of motile sperms are needed). Additionally, these spermatozoa also could prove advantage for conventional IVF or ICSI / fertility preservation where a large number of sperms are not required.

Gonadotrophin stimulation reduces follicular fluid hormone concentrations and disrupts their quantitative association with cumulus cell mRNA

Research questionDo follicular fluid hormone concentrations and the mRNA expression of LHCG, FSH and androgen receptors, aromatase and anti-Müllerian hormone (AMH) in cumulus granulosa cells differ in naturally matured and stimulated follicles? DesignCross-sectional study involving 57 natural cycle IVF (NC-IVF) and 36 conventional gonadotrophin-stimulated IVF (cIVF) cycles performed between 2014 and 2016. cIVF was performed by ovarian stimulation with human menopausal gonadotrophin and gonadotrophin-releasing hormone antagonists. Hormone concentrations were determined in the follicular fluid of the leading follicle, and mRNA concentrations were quantified by reverse transcription polymerase chain reaction in RNA extracted from granulosa cells of the cumulus oophorus complex obtained from these fluids. ResultsFollicular fluid hormone concentrations were significantly lower in cIVF compared with NC-IVF follicles. Median concentrations were 0.50 and 14.5 mIU/ml for LH (P < 0.001), 16.1 and 46.5 nmol/l for testosterone (P < 0.001), 1270 and 2675 nmol/l for oestradiol (P < 0.001), and 12.3 and 28.9 pmol/l for AMH (P < 0.001), respectively. In cumulus granulosa cells, mRNA concentrations for LH receptor, FSH receptor, androgen receptor, aromatase and AMH did not differ between cIVF and NC-IVF follicles. Several hormone and mRNA concentrations were quantitatively associated in natural cycles such as follicular fluid AMH and cumulus granulosa cells AMH RNA (r2 = 0.107, P = 0.013), follicular fluid testosterone and cumulus granulosa cell AMH RNA (r2 = 0.158, P = 0.002), follicular fluid oestradiol and cumulus granulosa cell AMH RNA (r2 = 0.105, P = 0.014) and follicular fluid oestradiol and aromatase (r2 = 0.113, P = 0.011). In contrast, these associations were not observed in stimulated cycles. ConclusionThese findings indicate some effects of gonadotrophin stimulation on follicular physiology, which could be a cause for the previously suggested lower oocyte quality in cIVF compared with NC-IVF.

P–248 Statistical estimation for incidence of blastocyst trophectoderm vesicles (TVs) and efficacy of assisted hatching (AH)

Abstract Study question The aim of this study is to analyse the association between blastocyst diameter and TVs development, and to examine the efficacy of AH. Summary answer Blastocysts with a diameter of more than 170 μm leads to high incidence of TVs and AH applied from the incidence should be effective. What is known already TVs are protrusion of trophectoderm cells often observed in expanding blastocyst stages. TVs can be observed in expanding blastocysts regardless of Intracytoplasmic sperm injection (ICSI) and Conventional-IVF (C-IVF), when the internal pressure of blastocysts increase. The rate of TVs incidence in blastocysts inseminated by ICSI is higher than that by C-IVF, due to penetration of the needle into the zona pellucida. Moreover, it has been reported that TVs may inhibit blastocyst hatching. However, the developmental timing of TVs is still unclear, and there is no study that has analysed the association between blastocyst diameter and the incidence of TVs. Study design, size, duration 1) Diameters and TVs incidence of blastocysts by ICSI and C-IVF were measured, and the cut-off value and the area under the curve (AUC) of the receiver operating characteristic (ROC) curve were calculated to estimate the timing of TV incidence. 2) We analysed the clinical pregnancy rates of blastocysts with TVs treated by AH compared to those of blastocysts by C-IVF not subjected to AH. Participants/materials, setting, methods This study included 821 transferred frozen blastocysts ranging from March 2018 to November 2019. The embryos were cultured in a dry incubator after insemination by ICSI or C-IVF. Blastocyst freezing conditions were set at day5 to day7 with a diameter of more than 150 μm in inner diameter of zona pellucida, and this was measured before freezing. The ROC curve was performed using EZR statistical analysis software. Main results and the role of chance 1) The incidence of TVs in blastocysts by ICSI and C-IVF was 27.5% (117/424) and 14.6% (58/397) respectively. The rate of the incidence of TVs in blastocysts inseminated by ICSI and C-IVF; 8.6% (12/140) and 0.95% (1/105) in 150–159 μm, 12.7% (14/110) and 8.2% (6/73) in 160–169 μm, 40.6% (28/69) and 10.5% (6/57) in 170–179 μm, 55.6% (30/54) and 25.5% (13/51) in 180–189 μm, 66.7% (20/30) and 35.7% (10/28) in 190–199 μm, and 68.4% (13/19) and 26.8% (22/82) in the diameter of more than 200 μm. The cut-off value of the ROC curve was respectively 170 μm (sensitivity 78.6% and specificity 73.0%) and 176 μm (sensitivity 84.5% and specificity 59.6%) in the diameter; the AUC was 0.8 [95%CI:0.752–0.848] and 0.74 [95%CI:0.687–0.793] respectively. 2) The clinical pregnancy rate of TVs blastocyst vs C-IVF blastocyst was 52.7% (88/167) vs 57.8% (37/64) respectively. There is no significant difference between the two clinical pregnancy rates (P = 0.556). Limitations, reasons for caution The findings of this study have to be seen in light of some limitations. Since this study aimed to analyse the incidence of TVs based on blastocyst size, we did not take into account the grade according to the Gardner classification and the number of trophectoderm cells. Wider implications of the findings: Blastocysts inseminated by ICSI and C-IVF were highly likely to have TVs above 170 μm and 176 μm respectively. The clinical pregnancy rates of the blastocyst with TV treated by AH was similar to those of the C-IVF blastocyst. Trial registration number Not applicable

P–257 An unknown cause lead to polyspermy in IVF cycles and 0PN zygotes in ICSI cycles in male patient

Abstract Study question The patient sperm has normal morphology and motility, which paternal factors cause the abnormal fertilization in IVF/ICSI and what is the underlying mechanism? Summary answer A genetic mutation of BEX1 and decreased PLC-zeta has been found in patient, which may provide novel insights of polyspermy and pronucleus formation during fertilization. What is known already In mammals, pronucleus formation, a landmark event for fertilization, is critical for embryonic development. Abnormal fertilization refers to the abnormal number of pronucleus and polar bodies in zygotes during in vitro fertilization, with an incidence of 5–15%, among which the incidence of polyspermy and 0PN is about 2–10% and 30%. However, the mechanisms underlying pronucleus formation still unclear. More research has focused on oocyte activation, while paternal relevant abnormal fertilizations have been rarely established. The mechanism of how sperm and/or substances carried by sperm influence the physiological process of fertilization is also unclear. Study design, size, duration In our study, we first work on the preliminary observation and analysis of sperm morphology, structure and sperm chromosome number, and then made further analysis at the genetic level to find out the cause of this particular phenotype in this patient. We use of zone-free golden hamster ova test the fertilizing capacity and rescue the pronucleus formation with SrCl2. Participants/materials, setting, methods The patient, golden hamster, Papanicolaou stain, scanning electron microscope (SEM), Transmission Electron Microscope (TEM), Fluorescence in situ hybridization (FISH), Whole Exome Sequencing (WES), IVF, ICSI, Assisted Oocyte Activation (AOA). Main results and the role of chance During 2016–2018, they did 4 cycle assistant reproduction technology. Cycle1, conventional IVF(C-IVF), 9 MII oocytes, 9 3PN zygotes; Cycle2, ICSI, 10 MII oocytes, 10 0PN zygotes; Cycle3, donor-oocytes C-IVF, 6 MII oocytes, 6 3PN zygotes, and the donor did C-IVF get normal zygotes and embryos; Cycle4, donor-sperm C-IVF, 7 MII oocytes, 4 2PN zygotes, 3 useable embryos. Remarkably, clinical examination about male shows normal sperm semen parameters. Papanicolaou stain and SEM shows that the sperm of the patient has normal morphology. The TEM data shows that the spermatozoa with normal head morphology and intact 9 + 2 sperm flagella structure. In the sperm FISH analysis, Chromosome ploidy is haploid. We performed WES on the male, after exclusion of frequent variants and application of technical and biological filters, two homozygous missense mutations were identified in BEX1 (c.191G&amp;gt;A [p. W64X]), which has been few reports of male infertility. The western blot result show that the PLC-zeta was decreased in patient. After 10mM SrCl2 assisted oocyte activation, the zygote has the pronucleus formation in ICSI. Limitations, reasons for caution At present, we only observe sperm related factors (morphology, structure, chromosome number, genetic mutation). Next step is to detect the substances sperm carried (e.g. RNA-seq, proteomics). In this case, what is of great concern to us is the inconsistencies of the abnormal fertilization during the conventional IVF and ICSI cycles. Wider implications of the findings: Many studies of fertilization mechanism, the main focus is on the maternal cytoplasmic factors, such as the Ca 2+ release initiate the fast block of oocytes. There are few reports about abnormal fertilization due to sperm factors. Our case may offer new insights for the study of fertilization. Trial registration number Not applicable

P–690 Clinical predictors of a high oocyte maturation rate in IVF treatment cycles

Abstract Study question Which clinical parameters predict a high oocyte maturation rate in patients undergoing IVF treatment? Summary answer Time between oocyte collection and insemination demonstrated significant association with oocyte maturation and represents a parameter that could be optimised in IVF cycles. What is known already Oocyte maturation is an important factor determining IVF outcomes and can be a rate-limiting step for patients undergoing treatment. A number of clinical and laboratory variables may affect this process, including the choice of trigger prior to oocyte collection, and certain laboratory procedures. Identification of which of these are predictors of maturation in individual centres enables local protocols to be optimised. Study design, size, duration This is a retrospective study of 714 oocyte collections from 661 women between January 2020 to November 2020 treated in a large, single centre in the UK. Subsequent fertilisation on fresh oocytes consisted of 371 IVF and 343 ICSI cycles. Participants/materials, setting, methods Patient and treatment data was collected by clinical staff at time of treatment. Either GnRH agonist, hCG or double trigger were administered 36 hours before collection. Prior to ICSI, oocyte maturation was assessed by visualisation of polar body (PB) extrusion. After IVF, the number of 2PNs plus unfertilised oocytes with PB extrusion were assessed. Univariate analyses consisted of Mann-Whitney test, t-test, Fisher’s Exact test or ANOVA. Potential predictors were investigated by logistic regression. Main results and the role of chance The end point was maturation rate, defined as high (greater or equal to 70%) or low (less than 70%). Factors predictive of a high rate included insemination more than 4 hours after collection. Oocytes inseminated over 4 hours post-collection displayed significantly higher maturation rates than oocytes inseminated less than 2 hours after collection (69% and 61% respectively; P = 0.01). Oocytes inseminated between 2–4 hours also had higher maturation than those inseminated less than 2 hours post-collection, but this did not reach significance (67% and 61%, respectively; P = 0.06). Further, oocytes fertilised by ICSI had significantly higher maturation than conventional IVF (77% and 67%, respectively, P &amp;lt; 0.001). No significant difference in oocyte maturation between triggers was observed. Similarly, neither age, AMH, a diagnosis of PCOS or number of oocytes collected predicted oocyte maturation in univariate analysis. Logistic regression analysis showed only time between oocyte collection and insemination (aOR 2.12; 95% CI 1.03–4.38; P = 0.04) to be a significant independent predictor. Limitations, reasons for caution Varying means of data collection across clinics and between clinical staff inevitably leads to provision of incomplete data and should be taken into consideration alongside interpretation. Prescription bias of specific triggers to certain patient demographics should be noted. Wider implications of the findings: Collectively, these results suggest that greater time between oocyte collection and insemination could be recommended to IVF clinics that wish to optimise their oocyte maturation. Triggering final maturation with GnRH agonist versus hCG or dual trigger did not have a significant effect on oocyte maturation when adjusted for confounders. Trial registration number Not applicable

P–064 Application of an artificial intelligence model for morphologic prediction of fertilization-competent human spermatozoa

Abstract Study question What is the role of artificial intelligence in selecting fertilization-competent human spermatozoa according to their morphological characteristics? Summary answer: The established AI model in this study can be potentially used to select semen samples with superior fertilization potential in clinical settings. What is known already Defective spermatozoa-zona pellucida (ZP) interaction causes subfertility and is a major cause of low IVF fertilization rates. While ICSI benefits patients with defective spermatozoa-ZP binding, a standard method to identify such patients prior to conventional IVF is lacking. The application of artificial intelligence to sperm morphology analysis has become a topic of growing interest owing to the fact that the conventional assessment is highly subjective and time-consuming. Deep-learning, a core element of artificial intelligence (AI), incorporates the convolutional neural networks (CNN) to process all the data composing a digital image through successive layers to identify the underlying pattern. Study design, size, duration The fertilization-competent spermatozoa were isolated according to their binding ability to the ZP. The ZP-bound and -unbound spermatozoa were collected for functional assays and to establish an AI model for morphologic prediction of sperm fertilization potential. Human spermatozoa (n = 289) were isolated from normozoospermic samples. Human oocytes (n = 562) were collected from an assisted reproduction program in Hong Kong. Sample collection has been ongoing and will continue until the end of this study in November 2021. Participants/materials, setting, methods Sperm-ZP binding assay was employed to collect ZP-bound and -unbound spermatozoa. The fertilization potential and genetic quality of the collected spermatozoa were evaluated by our established protocols. Diff-Quik- stained images of ZP-bound and -unbound spermatozoa were collected respectively for the establishment of an AI model. A novel algorithm for sperm image transformation and segmentation was developed to pre-process the images. CNN architecture was then applied on these pre-processed images for feature extraction and model training. Main results and the role of chance Our result showed that the sperm-ZP binding assay had no detrimental effect on sperm viability when compared with the raw samples and unbound-sperm subpopulations. ZP-bound spermatozoa were found with statistically higher acrosome reaction rates, improved DNA integrity, better morphology, lower protamine deficiency and higher methylation level when compared with the unbound spermatozoa. A deep-learning model was trained and validated by analyzing a total of 1,334 and 885 of ZP-bound/unbound spermatozoa to evaluate the predictive power of sperm morphology for ZP binding ability. Our newly trained AI-based model showed initial success in classifying the ZP-bound/ unbound spermatozoa according to their morphological characteristics with high accuracy of 85% and low computational complexity. Limitations, reasons for caution This sperm selection method requires micromanipulation and relatively long processing time to recover ZP-bound spermatozoa. In addition to limited availability, the use of human materials may result in interassay variations affecting the reproducibility of this method among laboratories. Wider implications of the findings: In light of current findings, AI-based sperm selection method may provide high predictive values of sperm fertilization potential for clinical purposes. This method is particularly applicable to patients who had poor fertilization outcomes after conventional IVF treatments or those with high degree of defective sperm-ZP binding ability. Trial registration number Not applicable

P-064 Application of an artificial intelligence model for morphologic prediction of fertilization-competent human spermatozoa

Abstract Study question What is the role of artificial intelligence in selecting fertilization-competent human spermatozoa according to their morphological characteristics? Summary answer The established AI model in this study can be potentially used to select semen samples with superior fertilization potential in clinical settings. What is known already Defective spermatozoa-zona pellucida (ZP) interaction causes subfertility and is a major cause of low IVF fertilization rates. While ICSI benefits patients with defective spermatozoa-ZP binding, a standard method to identify such patients prior to conventional IVF is lacking. The application of artificial intelligence to sperm morphology analysis has become a topic of growing interest owing to the fact that the conventional assessment is highly subjective and time-consuming. Deep-learning, a core element of artificial intelligence (AI), incorporates the convolutional neural networks (CNN) to process all the data composing a digital image through successive layers to identify the underlying pattern. Study design, size, duration The fertilization-competent spermatozoa were isolated according to their binding ability to the ZP. The ZP-bound and -unbound spermatozoa were collected for functional assays and to establish an AI model for morphologic prediction of sperm fertilization potential. Human spermatozoa (n = 289) were isolated from normozoospermic samples. Human oocytes (n = 562) were collected from an assisted reproduction program in Hong Kong. Sample collection has been ongoing and will continue until the end of this study in November 2021. Participants/materials, setting, methods Sperm-ZP binding assay was employed to collect ZP-bound and -unbound spermatozoa. The fertilization potential and genetic quality of the collected spermatozoa were evaluated by our established protocols. Diff-Quik- stained images of ZP-bound and -unbound spermatozoa were collected respectively for the establishment of an AI model. A novel algorithm for sperm image transformation and segmentation was developed to pre-process the images. CNN architecture was then applied on these pre-processed images for feature extraction and model training. Main results and the role of chance Our result showed that the sperm-ZP binding assay had no detrimental effect on sperm viability when compared with the raw samples and unbound-sperm subpopulations. ZP-bound spermatozoa were found with statistically higher acrosome reaction rates, improved DNA integrity, better morphology, lower protamine deficiency and higher methylation level when compared with the unbound spermatozoa. A deep-learning model was trained and validated by analyzing a total of 1,334 and 885 of ZP-bound/unbound spermatozoa to evaluate the predictive power of sperm morphology for ZP binding ability. Our newly trained AI-based model showed initial success in classifying the ZP-bound/ unbound spermatozoa according to their morphological characteristics with high accuracy of 85% and low computational complexity. Limitations, reasons for caution This sperm selection method requires micromanipulation and relatively long processing time to recover ZP-bound spermatozoa. In addition to limited availability, the use of human materials may result in interassay variations affecting the reproducibility of this method among laboratories. Wider implications of the findings In light of current findings, AI-based sperm selection method may provide high predictive values of sperm fertilization potential for clinical purposes. This method is particularly applicable to patients who had poor fertilization outcomes after conventional IVF treatments or those with high degree of defective sperm-ZP binding ability. Trial registration number not applicable

P–216 Successful pregnancies and deliveries in patients with a recurrent failure of ART treatments following artificial removal of the zona pellucida (ZP) at the pronuclear stage

Abstract Study question Can a novel embryo culture method that artificially removes the ZP at the pronuclear stage yield successful pregnancy in patients with poor-quality embryos and/or blastocysts? Summary answer A blastocyst transfer after ZP-free culture can result in pregnancy for patients who cannot obtain good quality blastocysts from conventional culture methods. What is known already Perivitelline threads are been associated with the formation of cytoplasmic fragments. We had previously observed perivitelline threads in the adhesive region between the ooplasm and the ZP at the first cleavage in human embryos. We removed the ZP at the pronuclear stage in 71 abnormally fertilized oocytes (zygotes with three pronuclei), donated after conventional IVF (c-IVF), and termed them ZP-free 3PN. We found ZP-free 3PN embryos could be cultured without losing blastomere adhesions. Furthermore, the rate of good quality embryos was significantly higher in ZP-free 3PN embryos compared with ZP-intact embryos (ZP-intact 2PN/2PB and 3PN embryos; P &amp;lt; 0.05). Study design, size, duration This study was conducted in two cases selected among patients who underwent ART treatment in our clinic between 2018 and 2019. Cases were selected if they lacked good quality blastocysts in previous c-IVF/Intracytoplasmic Sperm Injection (ICSI) cycles due to massive cytoplasmic fragmentation at the first and second cleavage. We performed a clinical trial of ZP-free culture from December 2019 to March 2020. Participants/materials, setting, methods Two cases were selected for this trial. Normally fertilized oocytes were grouped as ZP-free or ZP-intact. For the ZP-free group, 2PN embryos were placed in 0.125M sucrose-containing HEPES to reduce ooplasm size, then ooplasms were completely separated from ZPs by a laser and pipetting. ZP-free and ZP-intact embryos were cultured with time-lapse imaging for up to seven days. Resultant blastocysts were either transferred into uterus or cryopreserved on Day5/6/7 for future embryo transfer cycles. Main results and the role of chance The ZP-free culture method was applied to two patients (patient A and B) with recurrent failure of ART in our clinic due to poor-quality embryos and/or difficulties in obtaining good quality blastocysts. In both cases, blastocysts were successfully obtained and cryopreserved for all ZP-free culture cycles. In patient A, one good quality ZP-free blastocyst was freshly transferred five days after oocyte retrieval, and a live male baby (2925g) was delivered at 40 weeks of gestation by caesarean section). In patient B, a frozen/thawed ZP-free blastocyst transfer was conducted, and a live female baby (3225g) was delivered at 39 weeks of gestation by vaginal delivery. This shows ZP-free culturing may help obtain viable embryos in patients for which conventional in vitro culturing methods result in embryos characterized with severe cytoplasmic fragmentation and poor quality in the early cleavage stage. Limitations, reasons for caution Although successful pregnancies and deliveries were confirmed in two cases, postnatal evaluations will be absolutely necessary for infants derived from ZP-free culture. In addition, the number of trial cases needs to be expanded, however careful selection of suitable patients is necessary for this novel culture method. Wider implications of the findings: We found removing the ZP at the pronuclear stage improved embryo development and led to successful pregnancies and deliveries after blastocyst transfer. This indicates ZP-free culturing may be an effective method for decreasing cytoplasmic fragmentation caused by perivitelline threads or adhesion between the ooplasm and the zona pellucida. Trial registration number Not applicable