In a previous study, assisted sperm fusion insemination (ASFI), which is a fertilization method using a single motile sperm without breaking the oocyte membrane, yielded fertilization rates and blastocyst formation rates similar to those obtained by rescue intracytoplasmic sperm injection (ICSI) (1). In this study, we assessed embryo development following ASFI and rescue ICSI via a time-lapse system. A retrospective observational study using a time-lapse system was used to monitor 33 blastocysts from 25 patients undergoing ASFI (N=13 blastocysts) or rescue ICSI (N=20 blastocysts) between April 2019 and March 2020. Three hours after conventional in vitro fertilization, all inseminated oocytes were denuded of cumulus cells and the presence of the second polar body was confirmed. Oocytes with a single polar body were incubated for another 3 hours. Oocytes exhibiting only one polar body were defined as unfertilized oocytes and used for ASFI or rescue ICSI. Rescue ICSI was performed following conventional ICSI procedures. For ASFI, a motile sperm bound to the zona pellucida was collected using an injection needle and this sperm was pressed onto the membrane of an unfertilized oocyte for 30 seconds. Following fertilization, the oocytes were cultured and imaged using a time-lapse system. The mean and standard deviation of ages of the women in the ASFI and rescue ICSI groups were 37.3±4.0 and 35.3±4.4 years, respectively, which was not significantly different (p>0.05). The ASFI and rescue ICSI groups showed no statistical difference in the time of pronuclei appearance (4.8±1.1 vs. 5.2±1.2 hours), pronuclear fading (20.9±2.3 vs. 21.1±2.3 hours), formation of eight-cell stage (54.4±7.4 vs. 56.7±8.1 hours), initiation of blastulation (92.1±8.1 vs. 95.4±7.1 hours), and full blastulation (100.6±7.9 vs. 104.1±7.9 hours). This observational study showed that there were no differences in the morphokinetic parameters of embryo development between ASFI and rescue ICSI. For more general applications, further studies using a larger number of patients are necessary.

Purpose Chromosomal abnormalities are a major cause of spontaneous abortion, and conventional G-banded karyotyping (G-banding) is mainly utilized for chromosomal analysis. Recently, next-generation sequencing (NGS) has been introduced for chromosomal analysis. Here, we aimed to investigate the applicability and utility of NGS-based chromosomal analysis of products of conception (POC) on chorionic villus samples from spontaneous abortion. Methods The results of chromosomal analysis of 7 chorionic villus samples from spontaneous abortion were compared between conventional G-banding and NGS-based chromosomal copy number analysis. Age dependency and frequency of each chromosomal aneuploidy were evaluated for 279 cases analyzed by NGS. Results Excluding two cases (culture failure and maternal cell contamination), the results were consistent between G-banding and NGS. For cases analyzed by NGS, the rate of chromosomal abnormality increased in a maternal age-dependent manner. The frequency of each chromosomal aneuploidy detected by NGS was almost the same as that previously reported. Finally, NGS analysis was possible for difficult cases by G-banding analysis, such as culture failure, maternal cell contamination, long-term storage cases, and low cell number. Conclusions Chromosome analysis using NGS not only obtains comparable results to conventional G-banding, but also can analyze POC more accurately and efficiently.

Purpose To report a live birth after transfer of a vitrified-warmed blastocyst produced by assisted sperm fusion insemination (ASFI). Methods Oocyte retrieval and in vitro fertilization (IVF) were performed on a 37-year-old woman. Six hours after IVF, an oocyte exhibited a single polar body and so was defined as an unfertilized oocyte. A motile sperm was collected from the zona pellucida of the unfertilized oocyte by an injection needle. The motile sperm was pressed onto the membrane of the unfertilized oocyte. Results Two oocytes were matured and subjected to IVF. One of the 2 oocytes exhibited only one polar body and was defined as an unfertilized oocyte at 6 h after IVF; this oocyte then was subjected to ASFI. Two pronuclei were observed on the next day and cultured to the blastocyst stage. This embryo achieved blastocyst status and was vitrified on day 5. The resulting vitrified-warmed blastocyst was transferred, resulting in pregnancy and subsequent delivery of a healthy boy. Conclusion This report describes the first case of a successful birth following transfer of a vitrified-warmed blastocyst produced by ASFI.

This study aimed to determine whether fertilization can be obtained by assisted fusion of oocyte and sperm without breaking the oocyte membrane. A total of 79 infertile couples, each with at least one unfertilized oocyte after in vitro fertilization (IVF), were recruited. Sperm collected from the zona pellucida (ZP) were pressed onto the membrane of unfertilized oocytes at either 6 h or 24 h after IVF, a procedure that we designated as assisted sperm fusion insemination (ASFI). The results of ASFI were compared with those obtained in a previous trial on oocytes in which rescue intracytoplasmic sperm injection (ICSI) was performed at 6 h after IVF. Acrosome reaction (AR) rate of sperm bound to ZP, fertilization rate, degeneration rate, and blastocyst formation rate were evaluated. The AR rate of sperm collected from the ZP was significantly higher than that of the motile sperm recovered from around the oocytes but not bound to the ZP after IVF (98.0% vs. 28.6%). ASFI which was performed at 6 h after IVF yielded a mean fertilization rate of 73.4% (58/79), a degeneration rate of 0% (0/79) and a blastocyst formation rate of 60.8% (31/51). Rescue ICSI which was performed at 6 h after IVF yielded a mean fertilization rate of 70.0% (70/100), a degeneration rate of 4% (4/100) and a blastocyst formation rate of 42.4% (25/59). Binding of sperm to the ZP typically results in AR. ASFI with acrosome-reacted sperm collected from the ZP yielded the fertilization rates similar to those obtained with rescue ICSI.

It has been reported that the blastocyst formation rate of monopronuclear (1PN) embryos was significantly lower than that of two nuclear (2PN) embryos (especially in ICSI). However, a recent study revealed that 1PN embryos contained normal chromosome copy numberssimilar to those of 2PN embryos by preimplantation genetic testing for aneuploidy(PGT-A). We assessedeuploidy rate of 1PN embryosderived from ICSI with testicular sperm (TESE-ICSI) comparing to ejaculated sperm-ICSI or IVFby chromosomal analysis with next generation sequencing (NGS). Retrospective study in a single infertility center Patients who obtained1PN embryos were asked to participate in this study, and gave written informed consent for the study between April 2016 and March 2019. All 1PN cleaved embryos were cultured until the blastocyst stage followed by chromosomal analysis withNGS. Rates of 1PN, blastulation and euploidy were compared among three groups. The number ofembryos reached the blastocyst stage were 5 from TESE-ICSI, 42 from IVF and 19 from ejaculated sperm-ICSI, respectively. The average maternalage of couplesprovided embryos was 40.5 years. The Chi-squared testswere performed for statistical analysis and Pvalues less than 0.05 was defined as statisticaly significant. The incidence of 1PN embryos by TESE-ICSI (7.7%, 507/6562) was significantly (P<0.001) higher than that of IVF (3.8%, 845/22118) and that ofejaculated sperm-ICSI (3.7%, 1268/34547), while 2PN rate of ejaculated sperm-ICSI (72.3%) was significantly (P<0.001) higher than that of IVF (66.8%) andTESE-ICSI (51.4%). Blastocyst formation rate of 1PN embryo was 25.3% (173/684) in IVF, which was significantly (P<0.001) higher than that of ejaculated sperm-ICSI(11.4%, 110/965) and ofTESE-ICSI (12.2%, 51/417).Chromosomal analysis by NGScouldsuccessfully be performedin 1PN blastocystswith similar rate among three groups; 100% (42/42) in IVF, 94.1% (19/20) in ejaculated sperm-ICSI,and 100% (5/5) in TESE-ICSI. The euploidy rates of 1PN blastocystswere 21.4% (9/42) in IVF, 31.6% (6/19) inejaculated sperm-ICSIand 100.0% (5/5) in TESE-ICSI. Of all 1PN blastocysts, rate offemale embryos was significantly (P<0.05) higher (54.5%, 36/66) than that ofmale embryos (45.5%, 30/66). The male/female embryo ratio was 22/20 in IVF, 8/11 in ejaculated sperm-ICSI, and 0/5 in TESE-ICSI, respectively. TESE-ICSI generated significantly higher incidence of 1PN embryos than ejaculated sperm-ICSI, however the euploidy rate of 1PN blastocysts in TESE-ICSI was extremely high.Since we did not perform any tests to track biparental inheritance of the 1PN embryos, uniparental diploidy owing to endoreduplication of a haploid oocyte cannot be excluded. We, for the first time, revealedthat TESE-ICSI derived 1PN blastocysts barean extremely high euploidy rate. Although several mechanisms have been proposed to account for normal 1PN embryos, we speculate premature pronuclear breakdown by immature testicular sperm might occur during the first embryonic cleavage.

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.

Chromosomal abnormalities are the major cause of early pregnancy loss. Chromosome testing of products of conception (POC) provides valuable information for counseling and clinical managing of patients. We previously showed that next generation sequencing (NGS) can be utilized as a technique demanding lesser specimen with a lower failure rate, higher resolution, and shorter turnaround time than conventional karyotyping which is requiring labor-intensive and time-consuming cell culture with possible maternal cell contamination. We aimed to assess the efficacy of NGS method for chromosomal analysis of POC. In addition, we attempted to identify any associations between the incidence of chromosomal abnormalities and the profile of patients as well as fetal development in an assisted reproductive technology (ART) program. Retrospective study with a single reference genetic laboratory. Total of 131 consenting patients with first trimester miscarriages after vitrified-warmed embryo transfer were involved. POC samples were obtained bydilation and curettage between 7 to 10 gestational weeks. Chorionic villi were isolated under a dissecting microscopy, subsequently processed for NGS chromosomal analysis. Incidence of each chromosomal abnormality was reported and evaluated according to the patient profile, such as maternal age, previous history of miscarriage and fetal development. Finally, frequency of mosaics was also assessed. After NGS analysis, 28 cases (21.4%) were found to be normal, and the remaining 103 (78.6%) were abnormal, including 10 (7.6%) mosaics. Among normal karyotypes, ratio of female to male was 1.15 (15/13). Trisomies were the most common abnormalities except for the chromosome X monosomy (10.7%). Aneuploidy of chromosome 22 (20/113, 17.7%), 15 (16/113, 14.2%), 16 (16/113, 14.2%), X (13/113, 11.5%) and 21 (11/113, 9.7%) including overlaps, were most frequently involved. Mean maternal age of chromosomally abnormal cases was significantly higher than that of normal karyotypes (39.0 ± 18.5 vs 36.9 ± 16.5 years, P<0.05). Patients with more than equal 3 previous miscarriages showed a significantly lower rate of abnormalities than those with <3 miscarriages (28.6% vs 81.5%, P<0.01). Rate of abnormalities with positive fetal cardiac activity was not different from that of anembryonic pregnancies (80.0% vs 76.1%), although fetal cardiac activity was detected in all the 45,XO cases. Interestingly, however, mosaic abnormalities were significantly more often detected in anembryonic pregnancies than the other (15.2% vs 3.5%, P<0.05). With more conclusive and accurate results and higher resolution by NGS, we were able to characterize early pregnancy loss after ART, demonstrating relatively high rate of abnormalities with gender ratio being close to 1. Patients with repeated pregnancy loss showed lower chromosomal abnormalities indicating other causes for miscarriages in this group of patients. A higher incidence of mosaics detected in anembryonic pregnancies warrants further investigation.

AbstractPurposeTo evaluate the oocyte fertilization ability and embryo growth after cyclophosphamide (CPA) treatment in mice.MethodsMice were treated with CPA at different doses (0‐800 mg/kg body weight). The oocytes then were retrieved and evaluated for their in vitro fertilization efficiency.ResultsThe average number of metaphase II (MII) oocytes significantly decreased by ≥400 mg/kg CPA administration. The fertilization rate also decreased in the group that was treated with ≥400 mg/kg CPA. However, after fertilization, the embryos demonstrated normal growth ability. Two weeks after CPA administration, the number of mice from which the oocytes could be retrieved markedly decreased, but the fertilization rate and development of morphological features in the embryos were similar to those of the controls. One month after CPA administration, the number of mice from which the oocytes could be retrieved, fertilization rate, and development of the morphological features in the embryos were similar to those of the controls.ConclusionThe number of oocytes decreased as the CPA administration level increased; however, the oocytes' potential for fertilization and development to the blastocyst stage was not significantly affected. One month after CPA administration, the number of oocytes and the potential for development into blastocysts were recovered.

This study tried to cryopreserve low numbers of spermatozoa from men undergoing infertility treatments by inserting into agarose capsules. The capsules were transferred into a drop of cryoprotectant solution and injected 3–4 motile spermatozoa that were selected by the swim-up method by conventional intracytoplasmic sperm injection. These capsules were put on a Cryotop® and frozen in liquid nitrogen vapor, and then submerged into liquid nitrogen and subsequently thawed and recovered. The motile spermatozoa in the capsules were counted. Eventually, we cryopreserved 2142 motile spermatozoa in 702 agarose capsules from 26 male patients and 1356 (63%) spermatozoa maintained their motility after thawing. The spermatozoa motility rates after thawing (MRAT) ranged from 20.0% (5/25) to 95.1% (58/61) among patients. The median MRAT was 68.3% (interquartile range 46.1–75.7). The total number of motile spermatozoa collected by swim-up method strongly correlated with MRAT (r = 0.746). It was possible to cryopreserve spermatozoa from male patients undergoing infertility treatment using agarose capsules. However, there were wide differences in MRAT among patients. It seems the spermatozoa from semen where there were many motile spermatozoa may have higher freezing resistance. Further studies using this method in cryptozoospermic semen, testicular and epididymal spermatozoa are required.

Case: To present an extremely rare case of bilateral tubal pregnancies following a single-embryo transfer in a woman with a 4 year history of infertility prior to seeking assisted reproductive technology.Outcome: A pregnancy resulted from the transfer of an embryo that had been thawed from a frozen blastocyst during a hormone replacement cycle. An ultrasound that was performed at 5 weeks and 5 days of gestation revealed a gestational sac, embryo, and heartbeat in the right fallopian tube and similar signs of a gestational sac in the left fallopian tube. A laparoscopy revealed clear signs of an ectopic pregnancy in the ampulla of the right fallopian tube. Signs of swelling also were seen in the ampulla of the left fallopian tube. As the possibility of bilateral tubal pregnancies could not be ruled out, both fallopian tubes were removed. Pathological tests revealed chorionic villi and trophoblasts in both the left and right fallopian tubes.Conclusion: All previously reported cases of bilateral tubal pregnancies have been a result of multiple ovulations or multiple-embryo transfer and no case of bilateral tubal pregnancies after a single-embryo transfer has ever been reported. No genetic testing was performed; thus, it cannot be definitively stated that the divided chorionic villi and trophoblasts came from only one embryo.

Trophectoderm vesicles (TVs) are observed in some blastocysts that penetrate cells from the zona pellucida to the outer margin. Therefore, we compared this incidence in relation to hatching, pregnancy, and miscarriage rates between conventional in vitro fertilization (c-IVF) and intracytoplasmic sperm injection (ICSI). Vitrified/warmed blastocysts (n = 112) were derived from surplus embryos. The blastocysts were then observed using time-lapse cinematography to resolve the relationship between hatching and implantation. Another study was conducted that comprised 681 embryo transfer cycles in 533 patients who received a single vitrified/warmed blastocyst from our clinic. The incidence of TV was significantly higher in embryos inseminated by ICSI compared with c-IVF [ICSI: 51/56 (91 %); c-IVF: 25/56 (45 %); P < 0.01]. The successful hatching rate was significantly lower in ICSI than in c-IVF [ICSI: 11/56 (20 %); c-IVF: 29/56 (52 %); P < 0.01]. In addition, the hatching rate was significantly lower when TVs were present (14/76; 18 %) than in non-TV embryos (26/36; 72 %) (P < 0.01). In regard to the clinical study results, no significant differences were found between the groups in the pregnancy rate (TV present group: 107/183, 58.5 %; TV absent group: 273/498, 54.8 %) and miscarriage rate (TV present group: 21/107, 19.6 %; TV absent group: 53/273, 19.4 %). In vivo, we hypothesized that hatching and hatched would occur naturally by assisting protease action in the uterus; therefore, these results suggest that the presence of TV has no effect on pregnancy rates in the clinical setting.