Abstract Study question Can artificial removal of the zona pellucida (ZP-free) in patients whose embryos had severe fragmentation improve culture and pregnancy outcomes? Summary answer In patients with severe fragmentation, blastulation and live birth rate were improved by ZP-free procedure, and 14 live babieshave been obtained so far. What is known already In 2020, Yumoto et al. first reported that ZP-free procedure at the pronuclear stage decreased the rate of fragmentation and improved blastulation of 3 pronuclei embryos. Therefore, the ZP may not always be necessary for normal embryonic development after the pronuclear stage. The same group presented at the 37th ESHRE meeting in 2021, that the transfer of blastocyst derived from 2 pronuclei embryos (2PN) after ZP-free procedure resulted in normal pregnancy and live births in two patients who had had poor-quality embryos only before ZP-free procedure. We also presented culture outcomes improved by ZP-free procedure at the 2023 ESHRE meeting. Study design, size, duration This study included 571 embryos derived from 2PN on 118 cycles in 61 couples obtained between July 2021 and November 2023, whose previous oocyte retrieval cycle had shown massive cytoplasmic fragmentation at the first cleavage confirmed by a time-laps incubator (TLI) in two institutes. Mean age of women was 40.0±3.8 years. Culture and pregnancy outcomes after blastocyst transfer were compared with 1272 embryos derived from 2PN in the past 247 cycles without ZP-free procedure (ZP-intact). Participants/materials, setting, methods All participants provided written informed consent, and IRB approval was obtained. Embryos were placed in 0.125M sucrose-containing HEPES for cytoplasm shrinking at the pronuclear stage. The ZP was partially removed in non-adhesive areas with a laser system. Ooplasms were completely separated from ZP by blowing the medium to adhesion point like a jet car wash. The ZP-free embryos were cultured in TLI up to 7 days, followed by blastocyst cryopreservation for future embryo transfer cycles. Main results and the role of chance Day 3 embryos were divided into 3 groups using the Veeck’s classification: 8 cell embryos with grade 2 or higher were defined as good, embryos with grade 4 or 5 regardless of the number of cells were defined as poor, and others were defined as fair. We compared the culture outcomes of ZP-free cycle and previous ZP-intact cycle. For day 3 embryos, good, fair and poor grade were 15.6%, 55.3% and 29.1% in ZP-free embryos compared with 3.7%, 42.5%, and 53.8% in ZP-intact embryos, respectively (p < 0.001). As to the blastocysts, blastocysts development and good-quality blastocysts (G3BB or more on day 5 by the Gardner’s classification) rates were 36.9% and 19.4% in ZP-free embryos compared with 23.1% and 6.0% in ZP-intact embryos, respectively (p < 0.001). Clinical pregnancy rates for ZP-free and ZP-intact blastocyst transfer were 36.1% (22/61) and 15.0% (17/113), miscarriage rates were 18.2% (4/22) and 82.4% (14/17), and ongoing pregnancy rates were 29.5% (18/61) and 2.7% (3/113), respectively, with statistical significance after ZPF procedure (P < 0.01). Fourteen live babies have given birth by single blastocyst transfer with ZP-free procedure so far. Limitations, reasons for caution This study was retrospective study without any control group and was conducted at only two fertility centers. It is necessary to conduct sibling oocytes study or extensive randomized controlled trial to create criteria for patients who are benefit from ZP-free procedure. Moreover, safety of ZP-free culture should be evaluated. Wider implications of the findings ZP-free procedure in patients with severe fragmentated embryos at the pronuclear stage, reduced fragmentation and improved blastocyst development and clinical outcomes. We suggest that ZP-free embryos could be proposed as a new treatment option for patients who have had poor quality embryos with severe fragmentation. Trial registration number Not applicable

Are there any differences on clinical outcomes between two types of direct cleavage at the first division (DC1) in human embryos observed by time-lapse imaging? Blastocyst development of DC1 without any two-cell stage was lower than DC1 with momentary two-cell stage. Once they became blastocysts, they didn’t affect pregnancy outcomes. In recent years, time-lapse imaging (TLI) has been used to analyze dynamics on embryo development. It clarified that some embryo showed direct cleavage, where one cell divided into three or more cells directly (DC1). It has been reported that DC1 embryos showed chromosomal abnormalities and negative effect on the embryonic development compared to normal cleavage without DC1. Some reports revealed that once embryos with DC1 became blastocysts, they didn’t affect pregnancy outcomes compared to normal cleavage without DC1. No study has been published to date that categorizes DC1 into two types: DC1 embryos with or without momentary two-cell stage. This study included patients whose oocytes had been retrieved between January 2020 to December 2022. A retrospective study was conducted on 11,366 cleavage embryos derived from 2PN2PB in 2,735 cycles using time-lapse imaging (Geri®, Genea BIOMEDX, Australia, 5-minute intervals, 9 slices) after intracytoplasmic sperm injection (ICSI) with ejaculated spermatozoa. We focused on DC1 only and analyzed pregnancy outcomes on 248 cycles with single blastocyst transfer obtained from this study. Among 1,817 cycles where DC1 was observed by TLI, DC1 were classified into two types: DC1 embryos without any two-cell stage (DC1A) and with momentary two-cell stage (DC1B). We comparatively analyzed for blastocyst developmental rates, good-quality blastocyst (Grade 3BB or more by the Gardner’s classification) rates and pregnancy outcomes with single blastocyst transfer (clinical pregnancy rate, CPR; live birth rate, LBR). Statistical analysis was performed using student t-test and chi-square test. Among 2,735 cycles where ICSI was performed, female age was 39.3 ± 4.8 years (mean ± SD), previous ART cycles were 4.6 ± 5.5 times, and number of retrieved oocytes was 10.0 ± 8.7. DC1 embryos were found in 32.2% (3,663/11,366). Blastocyst development and good-quality blastocyst rates were 28.3% (826/2,921) and 14.6% (427/2,921) in DC1 embryos, whereas 67.5% (3,512/5,204) and 41.4% (2,157/5,204) in embryos without direct cleavage (non-DC), respectively. The former was significantly lower than the latter (P < 0.001). Among DC1 embryos, we confirmed DC1A was relatively fewer (21.2%) than DC1B (78.8%). Blastocyst development and good blastocyst rates were 11.7% and 2.1% in DC1A embryos, whereas 32.7% and 11.0% in DC1B embryos, respectively. The former was significantly lower than the latter (P < 0.001). CPRs, miscarriage rates and LBRs per single blastocyst transfer of DC1A were 44.4% (4/9), 11.1% (1/9), and 33.3% (3/9), while those of DC1B were 33.8% (47/139), 12.2% (17/139), and 21.6% (30/139), respectively. On the other hand, those of non-DC were 44.7% (232/519), 13.1% (68/519), and 30.6% (159/519). Both embryos with DC1A and DC1B produced healthy babies, except for one case with congenital hydronephrosis from DC1B. We excluded embryos with direct cleavage in the second division (DC2) in this study, because we focused on DC1 only. DC2 embryos may have complex effects besides DC1. The number of blastocysts from DC1A embryos may not be sufficient for comparison of pregnancy outcomes. Since blastocyst development and good-quality blastocyst rates were of DC1A was significantly lower than those of DC1B, it may be necessary to categorizes DC1 into two types: DC1 embryos with or without momentary two-cell stage. However, once they became blastocysts, they didn’t affect pregnancy outcomes. not applicable

Abstract Study question Can image analysis of ooplasmic fluidity in Piezo-ICSI predict unintentional membrane rupture (UMR) and subsequent oocyte degeneration? Summary answer Image analysis of ooplasmic fluidity using “optical flow” can be used to predict UMR and subsequent oocyte degeneration in Piezo-ICSI. What is known already UMR during Piezo-ICSI sometimes leads to oocyte degeneration, but not all oocytes degenerate. Leakage of ooplasm is often observed in degenerating oocytes after Piezo-ICSI. We speculated that the occurrence of ooplasmic leakage could relate to the ooplasmic fluidity. Optical flow is an image processing algorithm that analyses the movement of an object in a motion picture. In this study, the motion vectors for all pixels in the image were calculated using the dense optical flow method. Study design, size, duration We developed an image analysis system that detects the movement of the ooplasm on Piezo-ICSI videos using the OpenCV library. Videos of 96 oocytes recorded during Piezo ICSI (mean maternal age: 39.2±5.4 years) retrieved in our institute between January 2020 and September 2023 were retrospectively analyzed using the system. To minimize the potential influence of technical factors, Piezo-ICSI was performed by two embryologists. Participants/materials, setting, methods The oocytes were characterized by survival or degeneration and labeled with and without UMR. To evaluate the ooplasm fluidity, we analyzed the vertical motion vectors of all pixels between the start to puncture and the rupture of the oolemma (1st period) and between sperm injection and withdrawal of the pipette (2nd period). The velocity between groups was compared at the 95th, 99th, and 99.5th percentiles, followed by statistical analysis by the Mann-Whitney U test. Main results and the role of chance The 95th, 99th, and 99.5th percentiles of the survival group (n = 75) were significantly higher than those of the degeneration group (n = 21) in the 1st period (P < 0.01). Additionally, the 99th, and 99.5th percentiles of the survival group were significantly higher than those of the degenerative group in the 2nd period (P < 0.05). The 95th, 99th, and 99.5th percentiles without UMR (n = 52) were significantly higher than those with UMR (n = 44) in the 1st period (P < 0.001). Similarly, the 95th, 99th, and 99.5th percentiles without UMR were significantly higher than those with UMR in the 2nd period (P < 0.001). ROC curves were generated at the 95th, 99th, and 99.5th percentiles in the 1st period. The resultant areas under the curve (AUC) were 0.73, 0.75, and 0.73, respectively. Using a threshold of 0.75 for the 99th percentile, oocyte degeneration was predicted with a sensitivity of 85% and specificity of 57%. In the 2nd period, ROC curves at the same percentiles resulted in the AUCs of 0.63, 0.69, and 0.67, respectively. Using a threshold of 0.69 for the 99th percentile, oocyte degeneration was predictable with sensitivity as 57% and specificity as 86%. Limitations, reasons for caution This study did not control for variations among the embryologists performing Piezo-ICSI. The culture outcomes and clinical outcomes had not been evaluated. We only showed the relationship between ooplasmic fluidity and oocyte degeneration because it was a retrospective study. A prospective study using image analysis is needed. Wider implications of the findings Before the investigation, we expected that more ooplasmic fluidity (less viscosity) could increase UMR and oocyte degeneration. This study, however, indicated that more ooplasmic fluidity (less viscosity) decreased UMR and oocyte degeneration. Elucidation of the mechanism under this phenomenon may lead to reduced UMR and oocyte degeneration. Trial registration number Not Applicable

What is the risk factor of hypogonadism after micro TESE in NOA patients? Klinefelter syndrome (KS), past history of cryptorchidism, preoperative testicular volume <8 ml or testosterone <287 ng/dL are significant risk factors of hypogonadism after micro TESE. Micro TESE combined with intracytoplasmic sperm injection (ICSI) is currently standard procedure to treat infertility in cases of NOA. Although many studies have reported the effectiveness of the treatment concerning about sperm retrieval rates (SRR) and the live birth rates, data on safety, especially on the risk of hypogonadism, are limited. Most previous studies, including our past study, regarding the risk of hypogonadism after TESE have been limited to relatively few in number of patients and the investigations only of the changes of mean serum testosterone levels. This study retrospectively investigated 535 NOA patients including 336 cases of unexplained NOA, 91 KS, 16 other chromosomal abnormalities, 31 azoospermia factor (AZF)c deletions, 31 past history of cryptorchidism, and 30 post anticancer chemotherapy, who had undergone micro TESE at our institution from September 2013 to August 2018. All of them had been examined serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone levels before and at least once in 1-3 months after micro TESE. In this study, hypogonadism was defined as total testosterone <250 ng/dL, which is established by the Japanese Society of Men’s Health as diagnostic criteria for late onset hypogonadism syndrome, and/or starting testosterone replacement therapy (TRT). We assessed risk factors of hypogonadism after micro TESE using indicators such as age, preoperative testicular volume and testosterone levels, past medical history, unilateral or bilateral, first time or not micro TESE. In total of 535 men, FSH and LH after micro TESE significantly increased vs baseline (26.2 vs 29.9 IU/L, 9.6 vs 12.8 IU/L, respectively). On the other hand, testosterone levels significantly decreased after surgery (351.2 vs 297.5 ng/dL). Although postoperative hypogonadism was found in 42% (227/535), many of whom were cases of KS or preoperative testosterone levels <250 ng/dL. Sixteen men with KS, 3 men with unexplained NOA, and 2 men with past history of cryptorchidism started TRT because they complained clinical symptoms after micro TESE. Of the 21 men who started TRT, only 5 had new-onset low testosterone levels after micro TESE. According to the assessment of any association of various indicators and low postoperative total testosterone levels using multiple logistic regression analysis, significant risk factors of hypogonadism early after micro TESE were cases of KS (OR 11.4, 95% CI 4.4-29.9), preoperative testosterone levels <287 ng/dL (OR 7.5, 95% CI 4.7-12.0), preoperative testicular volume <8 ml (OR 3.9, 95% CI 2.3-6.4), past history of cryptorchidism (OR 2.9, 95% CI 1.1-7.8). Most examinations of postoperative hormone levels were performed in short-term period after micro TESE, so we could only assess the risk of postoperative hypogonadism in a short time course in this cohort study. The strength of the current study is that the risk factors of hypogonadism after micro TESE was assessed in a large population of NOA men with various etiologies. The results of this study provide useful clinical information about the risk of postoperative hypogonadism for NOA patients considering micro TESE. not applicable

Abstract Study question Does the combination of testicular sperm extraction (TESE) and ICSI increase the risk of embryo aneuploidy? Summary answer Embryo aneuploidy occurrence is not increased when using testicular sperm and is not affected by the etiology of azoospermia. What is known already TESE-ICSI, especially in patients with non-obstructive azoospermia (NOA), has been reported to result in poor outcomes and difficulty in obtaining good blastocysts, which may be due to immature sperm retrieved by TESE. In addition, patients who require TESE have been shown to be at particularly high risk of chromosomally abnormal sperm. These factors may increase the risk of embryo aneuploidy in TESE-ICSI and contribute to poor outcomes. However, few reports have investigated the results of PGT-A following TESE-ICSI. This study was therefore designed to investigate whether using testicular sperm is more likely to result in embryo aneuploidy. Study design, size, duration This retrospective study included 81 couples and 197 TESE-ICSI cycles undergoing PGT-A (average maternal age: 38.1±5.4 years) between September 2016 and November 2023. Of the total cycles, 94 cycles were case of obstructive azoospermia (OA) and 53 cycles were cases of NOA. The etiologies of NOA included were unexplained (22 cycles), Klinefelter’s syndrome (KS) (10 cycles), post-chemotherapy (3 cycles), post-orchiopexy (11 cycles), and microdeletion of azoospermia factor (AZF) c on the Y chromosome (7 cycles). Participants/materials, setting, methods ICSI outcomes were compared between different etiologies (OA vs. NOA). A total of 356 blastocysts acquired were analyzed using PGT-A with next-generation sequencing (NGS). The PGT-A results were classified according to maternal age (<38, 38-41, >41 years) and paternal age (<40, ≥40 years). In addition, TESE-ICSI cycles were limited to the maternal age of < 38 years to remove the effect of maternal factors, the euploidy rates were compared between different etiologies or morphological grade. Main results and the role of chance The rates of fertilization and blastocyst formation for TESE-ICSI were significantly lower in NOA than OA (47.4% vs. 61.6%, P < 0.001 and 37.3% vs. 50.7%, P < 0.01, respectively). The rate of good-quality blastocyst (16.6% vs. 21.2%) was also lower, but not statistically significant. The euploidy rate of all evaluated TESE-ICSI embryos decreased with increasing maternal age (52.9% in < 38, 29.3% in 38-41, 10.0% in > 41 years), but was not affected by the paternal age (40.6% in < 40, 42.5% in ≥ 40 years). For cycles with the maternal age of < 38 years, the euploidy rate was not significantly different between OA and NOA patients (55.3% vs. 49.3%). In addition, the association between embryo euploidy and morphological grade (using Gardner’s classification, ≥BB for good-quality vs. Limitations, reasons for caution Patients with chromosomal abnormalities, i.e. KS, could not be evaluated due to the small number of cases and patients with chromosomal translocation were not included in this study. Therefore, the effects of male factors in NOA on embryo aneuploidy require extended investigation. Wider implications of the findings In TESE-ICSI, especially in the case of NOA, it is difficult to obtain good quality embryos due to poor ICSI outcomes. However, if good quality embryos are obtained, the euploidy rate is high and there is no need to perform PGT-A on the sole basis of using testicular sperm. Trial registration number not applicable

Abstract Study question Are intravenous immunoglobulins (IVIG) effective for the selected patients who experienced unexplained recurrent pregnancy loss (RPL) that occurred at the same gestational weeks every time? Summary answer IVIG before and after the gestational week-limit (GWL), where miscarriage occurred at the same gestational weeks every time, may improve pregnancy outcomes in RPL patients. What is known already There is no established treatment for patients with unexplained RPL. Some investigators have indicated that IVIG may exhibit therapeutic efficacy in women with unexplained RPL. Since it has been reported that live birth rate significantly increased in women with four or more RPL of unexplained etiology, who received a high dose IVIG at 4-5 weeks of gestation in 2022, ESHRE Guideline Group on RPL (2022) has changed the recommendation to the following: the use of repeated and high doses of IVIG very early in pregnancy may improve live birth rate in women with four or more unexplained RPL. Study design, size, duration We performed a retrospective study between September 2013 to March 2023 in two fertility centers. We included 164 patients in the study who experienced 2 or more miscarriages between 5 and 21 weeks of gestation that occurred at the same gestational weeks every time, who had all negative results for our routine RPL work up and still unsuccessful result after treating these conditions, and whose products of conceptus (POC) revealed at least one normal karyotype. Participants/materials, setting, methods IVIG (Venoglobulin IH 5%, Japan Blood Products Organization, 0.4g/kg) injection was performed twice before and after 1 week of GWL. We divided 164 patients into IVIG group and non-IVIG group by their own choice. The primary outcome was live birth rate, and the secondary outcome was modified live birth rate excluding pregnancies with abnormal fetal karyotype, maternal and neonatal complications. All participants provided written informed consent, and Institutional Review Board approval was obtained. Main results and the role of chance IVIG group (n = 116) and non-IVIG group (n = 48) showed no significant differences in the background of patients on BMI (20.3±3.7 and 21.1±2.8), age (37.4±4.2 and 36.7±3.2) and Childbirth history (20.7% and 23.0%), respectively (average±SD), except for the history of past miscarriages. Average numbers of previous miscarriages on IVIG group and non-IVIG group were 3.2±1.3 and 2.5±0.8 (p < 0.001), respectively. After multivariate analysis for BMI, age, Childbirth history and previous miscarriages, live birth rate of IVIG group (70.7%, 82/116) was significantly higher than that of non-IVIG group (60.4%, 29/48; Odds ratio 3.40, 95% CI: 1.49-7.74, p = 0.003). Similarly, modified live birth rate of IVIG group (73.9%, 82/111) was significantly higher than that of non-IVIG group (63.0%, 29/46; Odds ratio 3.40, 95% CI: 1.49-7.74, p = 0.003). Treatment outcomes when IVIG was used for each number of previous miscarriages, the modified birth rate was 88.3% for two times miscarriages, 77.0% for three times, and 57.1% for four or more miscarriages. Maternal complications were observed in 2 cases in IVIG group (chest tightness, obstetric critical bleeding). There were no significant differences in the birth weight, gestational weeks, preterm birth, or fetal growth restriction of newborns between two groups. Limitations, reasons for caution Since retrospective nature and we did not perform POC tests for all patients whose pregnancies had ended in miscarriage, we could not assert the effectiveness of IVIG. We need to conduct randomized controlled trial using placebo group to demonstrate the IVIG efficacy before and after the GWL using euploid blastocysts. Wider implications of the findings IVIG before and after the GWL adjusted for each patient may improve pregnancy outcomes in RPL patients. Although IVIG is expensive, the current method requires only two IVIG injections, that may be acceptable for much more patients who want to avoid miscarriages and who didn’t use IVIG for economic reasons. Trial registration number NA

Abstract Study question Is the newly developed image analysis system capable of visualizing the shape features of the oolemma in real-time, useful for reducing oocyte degeneration during Piezo-ICSI? Summary answer Unintentional membrane rupture (UMR) can be reduced by this real-time image analysis, which lowers the risk of oocyte degeneration following Piezo-ICSI. What is known already UMR in the puncturing process of ICSI often predisposes oocyte degeneration. Identifying the appropriate puncturing positions may decrease the likelihood of UMR and thus degeneration, but this cannot be visualized via a microscope. We have reported that visualization of the shape features of the oolemma using moving image analysis during Piezo-ICSI can locate areas on the oolemma where UMR is likely to occur. It is feasible to assess the incidence of UMR and oocyte degeneration by performing ICSI while selecting appropriate puncturing positions with the aid of the newly developed imaging system. Study design, size, duration Our team have developed an image analysis system called ICSI POSITION DETECTOR (IPD), which can identify areas where rupture is likely to occur and visualize it in real-time using a video monitor. We prospectively evaluated the usefulness of IPD in a sibling oocyte study. From January 2020 to August 2021, a total of 1268 oocytes obtained in 225 oocyte retrieval cycles (average maternal age: 38.3 ± 4.7 years old) was included. Participants/materials, setting, methods The oocytes were randomly assigned to two groups: IPD-using vs. non-IPD-using. In the IPD-using group, Piezo-ICSI was performed at the “appropriate” position with a low chance of UMR indicated by IPD. In the non-IPD-using group, Piezo-ICSI was performed blindly. The rates of UMR, oocyte degeneration, fertilization and embryonic development were compared between the two groups. In addition, in the non-IPD-using group, moving images were recorded during Piezo-ICSI and analyzed retrospectively using IPD. Main results and the role of chance The rates of UMR and degeneration were significantly lower in the IPD-using group compared to the non-IPD-using group (6.0% vs. 11.9%, P < 0.001 and 1.6% vs. 4.6%, P < 0.01, respectively). The rates of fertilization (83.7% vs. 79.7%), blastocyst formation (51.9% vs. 51.0%), and good-quality blastocyst (24.7% vs. 24.3%) were not significantly different. Retrospective analysis of moving images using IPD on the non-IPD-using group showed that in 45.3% of oocytes (286 out of 632) ICSI was performed at a position with a high chance of UMR (inappropriate position). When ICSI was performed at the appropriate positions using IPD, the rates of UMR (6.0% vs. 18.2%, P < 0.001) and degeneration (1.6% vs. 7.3%, P < 0.001) were significantly lower, while the rates of fertilization (83.7% vs. 74.5%, P < 0.01) and blastocyst formation (51.9% vs. 43.0%, P < 0.05) were significantly higher than those when ICSI was performed at an inappropriate position. The rate of good-quality blastocyst (24.7% vs. 19.7%) was also higher, but not statistically significant. These results indicate that IPD can reduce the risk of UMR, thereby lower the degeneration rate. Furthermore, the embryonic development was better when using IPD to identify the appropriate position to perform ICSI, suggesting that UMR seems to be associated with poor ICSI outcome. Limitations, reasons for caution In this study, Piezo-ICSI was performed by only two designated embryologists in a single center. It did not assess clinical outcomes. Further research involving more embryologists is needed. Moreover, it would be feasible to investigate whether the usage of IPD is similarly effective on conventional-ICSI outcome. Wider implications of the findings The application of IPD to perform ICSI at the appropriate position can significantly avoid UMR, and thereby reduce oocyte degeneration and provide better embryonic development. We therefore, consider IPD to be a highly clinically useful tool, which contributes to the production of more embryos that can be used for treatment. Trial registration number not applicable

Abstract Study question Is it possible to identify the optimal puncture position by image analysis where oocyte degeneration is reduced in ICSI procedure? Summary answer Visualizing the likelihood of unintentional membrane rupture (UMR) using image analysis can reduce oocyte degeneration and thereby generate more embryos available for treatment. What is known already It is known that the oocyte degeneration after both conventional-ICSI and Piezo-ICSI are often observed when UMR occurs during the puncturing process and the likelihood of UMR may depends on the location of the puncture site on the oolemma. Identifying the appropriate puncturing position may decrease the likelihood of membrane rupture and thus degeneration, however, there are no studies identifying the optimal puncture position during ICSI. Study design, size, duration We have developed the ICSI Position Detector (IPD), which can identify an area where rupture is likely to occur and visualize it on a video monitor. This study included 1,110 mature oocytes retrieved from 190 consenting patients. Matured oocytes were inseminated either by Conventional-ICSI (n = 437, average maternal age: 39.1±4.6 y.o.) or Piezo-ICSI (n = 673, average maternal age: 38.6±4.5 y.o.). ICSI was performed blindly, moving images were recorded and analyzed retrospectively during ICSI using IPD. Participants/materials, setting, methods Inseminated oocytes were subsequently divided into two groups according to IPD results, oocytes on which ICSI was performed at the position with a low chance of UMR (appropriate group), and with a high chance of UMR (non-appropriate group) by IPD. The rates of UMR, degeneration, fertilization (2PN), blastocyst formation, and good-quality blastocyst (Grade 3BB and above on day 5 by the Gardner scoring) were compared between the two groups of both conventional-ICSI and Piezo-ICSI. Main results and the role of chance The areas in which UMR is less likely to occur and more likely to occur are heterogeneously distributed. When ICSI was performed blindly, about half was from the non-appropriate position. In appropriate group of Piezo-ICSI, rates of UMR (4.3% vs. 17.4%, P < 0.001) and degeneration (1.0% vs. 5.7%, P < 0.001) were significantly lower than those of non-appropriate group, whereas rates of fertilization (88.0% vs. 78.4%, P < 0.01) and blastocyst formation (57.8% vs. 45.9%, P < 0.01) were significantly higher than those of non-appropriate group, respectively. The rate of good-quality blastocyst (28.8% vs. 24.9%) was in favor of appropriate group, but not significantly different. In appropriate group of Conventional-ICSI, rates of UMR (6.7% vs. 20.6%, P < 0.001) and degeneration (1.5% vs. 6.6%, P < 0.01) were significantly lower than those of non-appropriate group, whereas rate of fertilization (88.7% vs. 69.5, P < 0.001) was significantly higher than those of non-appropriate group, respectively. The rates of blastocyst formation (37.5% vs. 35.3%), good-quality blastocyst (21.3% vs. 18.7%) were all in favor of appropriate group, but not significantly different. Limitations, reasons for caution This is a single private fertility clinic study. Its reproducibility should be assessed in different laboratory conditions and the hands of different operators. Moreover, specific studies should be addressed on the effect of the other putative confounders under investigation (e.g. kind of ovulation trigger, patient demographics, culture environment, etc.). Wider implications of the findings This study demonstrated that the IPD is useful to identify the optimal puncture location site to prevent UMR on ICSI procedure, resulting in reducing UMR and degeneration of the oocytes and increasing culture results, thereby, generating more embryos available for transfer or cryopreservation. Trial registration number not applicable

Abstract Study question To evaluate the effectiveness of chlormadinone acetate (CMA) for preventing premature LH surge in patients with normal ovarian reserve compared to cetrorelix. Summary answer In progestin-primed ovarian stimulation (PPOS) than GnRH antagonist (GnRH-ant), the incidence of premature LH surge was significantly lower, without significant difference in oocyte maturation rate. What is known already The GnRH-ant protocol is one of the conventional protocols which has some disadvantages including increased premature LH surge rate and cancelation rate. In recent years, the PPOS protocol has attracted attention as a new ovarian stimulation using progestin as an alternative to GnRH analog for suppressing a premature LH surge, however its efficacy is still controversial. In addition, many studies have investigated the reproductive outcomes of PPOS using medroxy-progesterone acetate or dydrogesterone; however, there are few reports of CMA, an oral progestin, which is inexpensive and widely used in Japan. Study design, size, duration This retrospective cohort study was performed in a reproduction center between March 2018 and October 2020 which included 977 Japanese patients with normal ovarian reserve undergoing PPOS with CMA (n = 299), or GnRH antagonist (GnRH-ant) with cetrorelix (n = 608) in their first IVF cycle at the reproduction center. In subgroup analysis, pregnancy outcomes after frozen embryo transfers (FET) between PPOS (n = 284) and GnRH-ant (n = 579) were also compared. Participants/materials, setting, methods The inclusion criteria were patients aged < 40 years and AMH ≧ 1.1 ng/mL, who underwent autologous oocyte retrieval in their first IVF cycle with freeze-all strategy. The primary outcome was the incidence of premature LH surge, the secondary outcomes was oocyte maturation rate. To reduce the impact of treatment bias and potential confounding factors, we conducted logistic regression models with inverse-probability-of-treatment weighting (IPTW). Main results and the role of chance After IPTW, baseline clinical data were well-balanced between the two groups, including age, AMH, BMI, the duration, type, and cause of infertility, antral follicle count, the history of recurrent spontaneous abortion, and previous IVF attempts. The premature LH surge rate was significantly lower with PPOS (3.1%) compared to GnRH-ant (20.1%) (odds ratio, 0.21; 95% confidence interval, 0.11–0.36). No significant differences were found in total gonadotropin dose (2400IU for PPOS vs 2400IU for GnRH-ant, p = 0.136), the number of oocyte retrieval (n = 15 vs n = 15, p = 0.484), oocyte maturation rate (78.8% vs 77.8%, p = 0.275), fertilization rate (73.0% vs 72.0%, p = 0.412), viable embryo rate per oocyte retrieval (40% vs 40%, p = 0.890), and good quality blastocyst rate (72.0% vs 69.6%, p = 0.092). However, the good quality day-3 embryo rate was significantly lower with PPOS (37.2% vs 49.1%, p < 0.05). There were no differences in the incidence of moderate-to-severe OHSS (0.3% vs 0.7%, p = 0.481). In FET cycles, the pregnancy outcomes, such as implantation rate (43.1 % vs 51.9 %, p = 0.013) and clinical pregnancy rate (46.5% vs 54.7%, p = 0.027) were significantly lower with PPOS, however, no significant differences were found in ongoing pregnancy rate (75.6% vs 80.5%, p = 0.325), and live birth rate (72.4% vs 79.5 %, p = 0.142). Limitations, reasons for caution This was a retrospective cohort study conducted in a single center. The participants in this study were limited to Japanese ethnicity. The results need to be validated across different centers and other ethnicities. Wider implications of the findings This is the first report assessing the reproductive outcomes on PPOS using CMA, widely used in Japan. The PPOS with CMA significantly suppressed the premature LH surge rate compared to GnRH-ant protocol, without decrease in oocyte maturation rate. Trial registration number N/A

Abstract Study question What is the frequency of azoospermia factor (AZF) microdeletions and sperm retrieval rate (SRR) by micro TESE in patients with these deletions? Summary answer AZFc is most frequent of Y chromosome microdeletions and a predictor of micro TESE outcome in Japanese azoospermic men. What is known already After Klinefelter syndrome, Y chromosome microdeletions are the second most frequent genetic cause of male infertility, with a prevalence of 2%-10% in non-obstructive azoospermia (NOA) and three spermatogenesis loci in the Y chromosome long arm (Yq11) have been classified as AZFa, AZFb, and AZFc. The classical correlation of histopathology phenotypes with these three microdeletions comprises of complete absence of germ cells (Sertoli cell-only syndrome) in patients with AZFa microdeletions, maturation arrest of meiosis in patients with AZFb microdeletions, and hypospermatogenesis in patients with AZFc microdeletions, however, individual variation in the extent of deletions has led to various spermatogenic phenotypes. Study design, size, duration We performed a retrospective study based on two reproduction centers in Japan and evaluated 1373 azoospermic patients in our clinics between September 2013 and December 2021. We investigated the frequency of AZF microdeletions and SRR by micro TESE in patients with these microdeletions and therefore aimed to evaluate the correlation between AZF microdeletions and micro TESE results. Participants/materials, setting, methods A total of 1373 azoospermic were enrolled. After the diagnosis of azoospermia, karyotype analysis and detection of Y chromosome microdeletions were performed on peripheral blood lymphocytes of these patients. Y chromosome microdeletions in AZFa, AZFb, and AZFc regions were detected using Promega Y Chromosome AZF Analysis System version 2.0 (Promega Co.). Twenty sequence-tagged sites within the AZF region of Yq11 and the sex-determining region Y gene were targeted for polymerase chain reaction (PCR) amplification. Main results and the role of chance One hundred and fifty-two AZF microdeletions (11.1%) were detected in the azoospermic patients. The most common deleted region was AZFc (60 cases, 4.4%). Among the patients, 17 (1.2%), 1 (0.1%), 42 (3.1%), 13 (1.0%), and 6 (0.5%) had AZFa, AZFa+b, AZFb+c, AZFb, and AZFa+b+c microdeletions, respectively. When the cases were grouped according to causes of infertility that could be detected, no Y chromosome microdeletions were detected in some groups (cases with Klinefelter Syndrome, hypogonadotropic hypogonadism, congenital absence of vas deferens, and 47, XYY karyotype). Fifty-three azoospermic men with AZFc microdeletions underwent micro TESE, and spermatozoa were detected in 88.7% (47/53) of these men. In contrast, we detected spermatozoa in only 20.4% (109/534) of the azoospermic men without AZF microdeletions. The SRR was much higher in patients with AZFc microdeletions than that of patients without AZF deletions. Although three azoospermic men with AZFb+c microdeletions had also undergone micro TESE following patient request, we did not retrieve spermatozoa. Limitations, reasons for caution We excluded post chemotherapy NOA showing 46, XX and AZFa+b+c deletions post bone marrow transplantation from female donor. Additionally, we did not detect AZFc partial deletion including gr/gr deletion. The cohort size of this study is not small, however, our screened population of infertile men may be biased. Wider implications of the findings NOA patients with AZFc microdeletions had a high percentage of successful sperm retrieval by micro TESE. Our study emphasizes that diagnosis of Y chromosome microdeletions is critical for preconception genetic counseling and provides clinically valuable prognostic information to couples considering surgical sperm retrieval. Trial registration number None

Abstract 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

Abstract Study question To analyze whether microfluidic sperm selection (MSS) by ZyMōt™ improves sperm DNA fragmentation rate and embryonic development compared to density gradient centrifugation with swim-up (DGCS). Summary answer MSS by ZyMōt™ selects sperm for clinical use with less DNA damage significantly compared to DGCS. What is known already Conventional sperm preparation methods, such as density gradient centrifugation and the swim-up method utilize centrifugation during processing, may damage the sperm. MSS may allow for improved selection of normal sperm compared with conventional sperm preparation as it yields sperm with a lower DNA fragmentation rate. However, there are few clinical studies by sibling oocytes study compared to DGCS. Study design, size, duration This prospective study was performed between March 2020 and May 2020 at a reproductive center. All patients involved gave written consent, and institutional review board approval was granted. A total of 575 metaphase II oocytes were collected from 49 cycles. Wife’s age was 34.7 ± 3.9 years old. Raw sperm concentration and motile sperm concentration was 63.1 ± 78.7M/mL, and 41.6 ± 67.7M/mL, respectively. Participants/materials, setting, methods Patients who performed ART for the first or second time were divided into two groups according to MSS and DGCS. Sperm DNA fragmentation rate (SDFR) and motile sperm concentration were compered between MSS and DGCS. SDFR was measured by sperm chromatin structure assay (SCSA) using a flow cytometer. Sibling oocytes were randomized into MSS-IVF, DGCS-IVF, MSS-ICSI, and DGCS-ICSI. Rate of two pronuclear (2PN) oocytes, blastocysts development, and good-quality blastocysts were compared between each group. Main results and the role of chance SDFR was 13.5 ± 11.8% for raw semen. SDFR was significantly lower after MSS (3.6 ± 4.1%) than that for raw semen and after DGCS (17.4 ± 14.8%) (P < 0.01). Motile sperm concentration after MSS (19.0 ± 28.3M/mL) was significantly higher after than after DGCS (15.4 ± 15.3M/mL) (P < 0.01). The number of IVF performed was 145 for MSS and 132 for DGCS. IVF results (MSS vs DGCS) were 2PN rate (73.1% vs 72.0%), blastocysts development rate (65.3% vs 55.4%), and good quality blastocysts rate (43.2% vs 34.9%). The number of ICSI performed was 149 for MSS and 149 for DGCS. ICSI results (MSS vs DGCS) were 2PN rate (77.9% vs 79.2%), blastocysts development rate (68.8% vs 65.8%), and good quality blastocysts rate (35.8% vs 30.6%). No significant difference was observed between MSS and DGCS for each parameter both IVF and ICSI. Limitations, reasons for caution The participants were limited to those who collected semen of 2mL or more and motile sperm concentration of above 1M/mL, because semen sample needed to be divided to MSS and DGCS. Wider implications of the findings: This is the first study to conducted in sibling oosytes study with MSS and DGCS, in both IVF and ICSI. MSS is effective in collecting sperm with less DNA damage compared to DGCS. Motile sperm concentration after using MSS is sufficient to perform IVF as well as DGCS. Trial registration number Not applicable

Introduction Chromosomal abnormalities are the most common reason for spontaneous abortion. Conventional cytogenetic analysis by G-banded karyotyping is generally performed for chromosomal analysis, but it has a problem with low-resolution, and is needed long term cell culture and enough experience for diagnosis. More recently, next generation sequencing has been introducing and improving for chromosomal analysis as an accurate, high resolution and throughput method. In this study we aimed to compare the consistency between conventional G-banding and NGS-based chromosomal copy number analysis for chorionic villus from spontaneous abortion. In addition, the frequency of each chromosomal aneuploidy was evaluated. Materials and methods From February, 2018, to April, 2018, chromosomal analysis for 7 chorionic villus samples from spontaneous abortions (from 7 to 9 weeks) were carried out both conventional G-banding and NGS (VeriSeq-PGS, Illumia). The frequency of each chromosomal aneuploidy was investigated for 110 cases from February, 2018 to December, 2018. Results NGS was able to analyze all 7 cases, but G-banding was able to detect 6 cases, and one case was cell growth failure. In 6 cases analyzed by G-banding, the results of 5 cases were consistent with the results of NGS, but one case suspected maternal cell contamination. Among 7 cases analyzed by the NGS, 2 cases were normal male karyotype (46, XY) and 5 cases were autosomal trisomy, implying that there were no cases suspected of maternal cell contamination. Among 110 cases, chorionic villus was not observed under microscope from 18 samples, NGS result was obtained from 92 cases. Seventy-one cases were found to have abnormal chromosomes (71/92, 77.2%), and 21 cases were normal karyotype (21/92, 22.8%). Aneuploidy of chromosome 22 (21.8%), chromosome 16 (17.9), chromosome 15 (11.9%), chromosome 21 (10.3%) and Chromosome X (10.3%) were more frequently, consistent with previous report. Conclusions Chromosome analysis using NGS not only obtained comparable results to conventional G-banding, but also is able to analyze more accurately and quickly.

Purpose of Investigation: The objective of this prospective controlled observational study was to evaluate the effect of myoinositol supplementation on intracytoplasmic sperm injection (ICSI) outcome in Japanese infertile polycystic ovarian syndrome (PCOS) women with non-obese less-androgenic phenotype, which is common in East Asia. Materials and Methods: The ICSI outcome in the first treatment cycle under a flexible gonadotropin-releasing hormone antagonist protocol was compared between 25 PCOS women taking 4 g/day myoinositol + 400 mu g/day folic acid (Myo/FA group) and 25 PCOS women taking 400 mu g/day FA alone (FA Group). Results: The total dosage of human menopausal gonadtropin was significantly lower in the Myo/FA group than in the FA group (p = 0.034). The number (p = 0.000029) and rate (relative risk 1.47, 95% confidence interval 1.43-1.50, p < 0.0001) of metaphase II oocytes and the number of fertilized oocytes (p = 0.0074) was significantly higher in the Myo/FA group than in the FA group. Conclusion: Myoinositol supplementation is a safe and effective treatment modality to increase the mature and fertilized oocytes, along with a reduction in the gonadotropin dose in ICSI cycles in Japanese infertile PCOS women with non-obese less-androgenic phenotype.