Chromosomal copy number analysis of products of conception by conventional karyotyping and next-generation sequencing.

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.

First birth following assisted sperm fusion insemination using sperm bound to zona pellucida.

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.

Fertilization with human sperm bound to zona pellucida by pressing onto the oocyte membrane.

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.

Effects of cyclophosphamide administration on the in vitro fertilization of mice.

PURPOSE: To evaluate the oocyte fertilization ability and embryo growth after cyclophosphamide (CPA) treatment in mice. METHODS: Mice 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. RESULTS: The 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. CONCLUSION: The 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.

Bilateral tubal pregnancies after a single-embryo transfer.

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.

Cryopreservation of very low numbers of spermatozoa from male patients undergoing infertility treatment using agarose capsules.

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.

Effect of the presence of trophectoderm vesicles on blastocyst in relation to in vitro hatching, clinical pregnancy, and miscarriage rates.

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.

Single human sperm cryopreservation method using hollow-core agarose capsules.

OBJECTIVE: To develop an efficient cryopreservation method using a single sperm. DESIGN: Experimental study. SETTING: Laboratory of a private institute. PATIENT(S): A fertile donor. INTERVENTION(S): We produced hollow-core capsules with agarose walls. A single human sperm was injected into each capsule as per the conventional intracytoplasmic sperm injection (ICSI) method. The capsules that contained the spermatozoa were cryopreserved on polycarbonate or nylon mesh sheets using nitrogen vapor. Before their use, the capsules were thawed and recovered. The motile spermatozoa in the capsules were counted. MAIN OUTCOME MEASURE(S): The recovery rates of the agarose capsules and the spermatozoa in these capsules after thawing and the mortality and survival rates of the spermatozoa. RESULT(S): The recovery rates of the capsules were 91.5% (75/82) using polycarbonate sheets (PS) and 98.3% (59/60) using mesh sheets (MS) after thawing. The recovered capsules were not at all damaged. The recovery rates of the spermatozoa were 91.5% (75/82) using PS and 96.7% (58/60) using MS. Sperm motility rates were 85.3% (64/75) and 82.8% (48/58), whereas the survival rates of the immotile spermatozoa by the hypoosmotic swelling test were 81.8% (9/11) and 50.0% (5/10); furthermore, the total survival rates of the spermatozoa were 97.3% (73/75) and 91.4% (53/58) using PS and MS, respectively. There was no significant difference between the results obtained using PS and MS. CONCLUSION(S): A cryopreservation method for a single sperm using an agarose capsule has been developed. The method is expected to be useful in ICSI treatment in patients with few spermatozoa.

In vitro fertilization embryo development from caffeine-treated murine sperm.

Purpose: To evaluate the effect of long-term caffeine administration on murine sperm and subsequent in vitro fertilization (IVF). Methods: Male mice were injected with various doses (0, 0.2 and 1.0 mg/mouse/day) of caffeine for 1 month. After sperm collection, the IVF rate and embryo development to the blastocyst stage were evaluated. Results: The mean body weight significantly decreased in the 1.0 mg/day treatment group compared to the control group (P < 0.01). Testicular weight and histological features did not differ, and total blood testosterone was no different in spite of the difference between 0.2 and 1.0 mg/day of caffeine. The IVF rate differed significantly between the control group [100/105 (95.2 %)] and 0.2 mg/day group [106/121 (87.6 %)] (P < 0.05). Furthermore, blastocyst formation was significantly and dose-dependently lower with higher caffeine levels: control group: 85/100 (85.0 %); 0.2 mg/day group: 84/106 (79.2 %) (P < 0.05); 1.0 mg/day group: 64/102 (62.7 %) (P < 0.001). Conclusions: Caffeine treatment affected body weight of male mice. However, testicular weight, histological features and total blood testosterone concentration were not statistically different. In addition, following IVF using sperm from these mice, blastocyst formation decreased in a dose-dependent manner. These findings suggest that embryo development from oocytes fertilized with sperm from caffeine-administered male mice is negatively affected.

Deliveries of babies with normal health derived from oocytes with smooth endoplasmic reticulum clusters.

PURPOSE: To examine the impact on development of derived embryos from smooth endoplasmic reticulum clusters (SERC) in human metaphase II (MII) oocytes. METHODS: Retrospective analysis at Kyono ART Clinic. Comparison of embryological development, pregnancy, live birth and fetal malformation between oocytes with SERC (the SERC(+) group) and those without (the SERC(-) group) in 2,158 patients (3,758 cycles) after ICSI. RESULTS: Fertilization and implantation rate were significantly lower in SERC(+) MII oocytes than in SERC(-) MII oocytes. After the transfer of fresh and vitrified embryos derived from SERC(+) oocytes, 14 pregnancies resulted in 14 healthy babies, including 2 from fresh embryo transfer (ET) and 12 from vitrified-warmed ET, with no malformations. CONCLUSION(S): The presence of SERC in MII oocytes was associated with significantly lower fertilization rates and implantation rates than seen in SERC(-) MII oocytes within SERC (+) cycles. However, SERC had no impact on post-implantation development as well as neonatal outcome.

Case report: a successful pregnancy outcome in a patient with non-mosaic Turner syndrome (45, X) via in vitro fertilization.

We describe a successful pregnancy outcome in a patient with non-mosaic Turner syndrome (45, X) via in vitro fertilization. The patient achieved a second pregnancy at 35 years of age. The her blood lymphocyte karyotype was examined by G-band and FISH. Furthermore, cumulus cells and her elbow skin cells were evaluated via FISH. Non-mosaic Turner syndrome was determined by G-banding [100 % (50/50) 45, X]. Lymphocytes were shown as 478/500 (95.6 %) cells of X sex chromosome signal, 15/500 (3.0 %) cells of XXX signal, and 7/500 (1.4 %) cells of XX signal. The cumulus cells were mosaic: 152/260 (58.5 %) were X; 84/260 (32.3 %) were XXX, 20/260 (7.7 %) were XX, and 4/260 (1.5 %) were XY. Moreover, skin cells included a mosaic karyotype [47, XXX(29)/46, XX(1)]. We conclude that the collection of a large number of blood lymphocytes can reveal different mosaic patterns (X, XX and XXX) by FISH in spite of non-mosaic Turner syndrome.

Effect of long-term caffeine administration to mice on in vitro fertilization and embryo development using oocytes.

PURPOSE: To evaluate the effect of long-term caffeine administration to mice on in vitro fertilization (IVF) of oocytes. METHODS: Mice were injected with different dosages (0, 0.1, and 1.0 mg/mouse/converted day) of caffeine for one month. Subsequently, the fertilization rate and embryo development to blastocyst stage were evaluated in IVF using oocytes from the mice. RESULTS: The retrieved average oocyte rate was significantly lower (27.4) in mice injected with 1.0 mg caffeine than in the control group (36.5; P < 0.05); the fertilization rate was significantly different between the 0 mg (317/401; 79.1 %) and 1.0 mg group (199/301; 66.1 %) (P < 0.05). At 96 h after insemination, the blastocyst formation rate was significantly decreased in the 1.0 mg group (94/199; 47.2 %) compared with the control (0 mg) group (237/317; 74.8 %) and 0.1 mg group (226/323; 70 %) (P < 0.05). When 1.0 mg caffeine was administered for two weeks, embryo development was significantly impacted. CONCLUSIONS: Our findings suggest that caffeine administration negatively impacts oocytogenesis and embryonic development after IVF.

Patients with 47, XXX karyotype who experienced premature ovarian failure (POF): two case reports.

PURPOSE: Pubertal onset and sexual development are usually normal in 47, XXX individuals; however, we report two cases of premature ovarian failure (POF) in infertile women with trisomy X. METHODS: Chromosome analysis was conducted with G-banding and fluorescence in situ hybridization using X- and Y-bearing probe. Hormonal administration was primarily Kaufmann's treatment or long-term estradiol treatment, followed by withdrawal bleeding from estrogen and progesterone. RESULTS: Two patients with trisomy X, aged 31 (patient 1) and 27 years (patient 2), were diagnosed with POF due to hypergonadotropic hypogonadism. Their ovaries were small. Patient 1 had a FSH level of 44.6 mIU/ml and patient 2 had a FSH level of 74.6 mIU/ml. In patient 1, with Kaufmann's treatment, the FSH decreased to 13.5 mIU/ml; however, follicle growth did not occur following HMG stimulation. In patient 2, FSH did not decrease despite Kaufmann's treatment; therefore, she was given a GnRH agonist and her FSH level decreased to 7.1 mIU/ml. However, her ovaries never responded to HMG stimulation. CONCLUSION: We report on two patients with a 47, XXX karyotype who became infertile due to POF. We recommend that when a patient is diagnosed with trisomy X, the possibility of POF must be strongly considered.

Successful second delivery outcome using refrozen thawed testicular sperm from an infertile male true hermaphrodite with a 46, XX/46, XY karyotype: case report.

We report a successful second delivery of a healthy infant fathered using refrozen thawed testicular sperm from an infertile male chimera. We also examined sex chromosome distribution of the seminiferous tubule. Intracytoplasmic sperm injection (ICSI) was performed using the remaining refrozen testicular sperm, which had been stored during the first treatment. Biopsied testicular cells were examined by fluorescence in situ hybridization (FISH) and the peripheral lymphocyte karyotype was tested using a G-band. Following ICSI, a second pregnancy was established, and a healthy girl was successfully delivered at 40 gestational weeks without complications. Although the husband's lymphocyte chromosomal analysis revealed a 46, XX [28]/46, XY [2] karyotype, the seminiferous tubule cells on histological examination by FISH were chimeric sex chromosome type XX [18]/XY [82]. In conclusion, this is a very rare case report of a successful subsequent delivery of a healthy infant (46, XX) from an infertile true hermaphrodite (46, XX/46, XY) using refrozen thawed testicular sperm. The seminiferous tubule cells' karyotype ratio differed from that of the lymphocytes.

Successful delivery following intracytoplasmic sperm injection with calcium ionophore A23187 oocyte activation in a partially globozoospermic patient.

PURPOSE: To describe a successful pregnancy outcome following intracytoplasmic sperm injection (ICSI) with assisted oocyte activation (AOA) in a case of partial globozoospermia. METHODS: AOA was accomplished with calcium ionophore A23187. Sperm morphology was observed via light, fluorescent and electron microscopy following a Diff-Quik stain and fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) staining. An activation ability test was employed using a mouse oocyte exposed to strontium chloride. RESULTS: Via light microscopy, it was found that a large number of sperm possessed deficient acrosomes and a sharply rounded head; however, we observed both normal and the aforementioned abnormal sperm via FITC-PNA staining of a semen specimen. Mouse oocyte activation was 87.5 % via natural activation without AOA. With AOA after ICSI, 100 % oocyte activation was observed. Five oocytes were retrieved, and AOA with A23187 after ICSI resulted in a high fertilization rate (4 of 5, 80 %). Two embryos developed and the patient subsequently delivered a healthy female infant without any congenital abnormalities. CONCLUSIONS: We report a successful pregnancy outcome using an early stage embryo, which developed following ICSI using sperm from a partially globozoospermic patient who possessed temporary potential oocyte activation.

Perinatal outcome of twice-frozen-thawed embryo transfers: a clinical follow-up study.

We evaluated our clinical data on refrozen-thawed ETs (92 cycles) and found that human embryos were capable of withstanding two freeze-thaw cycles, resulting in normal live births after transfer at a rate similar to that of primary frozen-thawed embryos. This is the first follow-up study to present perinatal outcomes of children born after embryo re-cryopreservation, and our results should encourage clinicians to explore the possibility of performing the refreezing procedure.

Proliferation of mouse spermatogonial stem cells in microdrop culture.

It is now possible to make mouse spermatogonial stem cells (SSCs) proliferate in vitro. However, these cultured cells, called germ-line stem (GS) cells, consist of not only SSCs but also a greater number of progenitor spermatogonia. Moreover, isolated GS cells barely proliferate. To elucidate the nature of SSCs and progenitor spermatogonia, we adapted a microdrop culture system to GS cells. Using a micromanipulator, individual microdrops were seeded with clusters or dissociated known numbers of GS cells. The number of surviving colonies was determined after 30 days. The proliferation rate of GS cells in microdrops increased as the number of GS cells seeded increased. It was observed that as few as three GS cells seeded in a microdrop can proliferate and expand the colony size. Those GS cells of expanded colonies were able to proliferate following subculture and underwent spermatogenesis in the host testis after transplantation into the seminiferous tubules of recipient mice. These data revealed that SSCs can multiply in a microdrop culture system. Microdrop culture offers a novel tool to elucidate the nature of SSCs in regard to their self-renewing capacity and can serve as a monitoring system of culture conditions for the self-renewal of SSCs.

In vitro murine spermatogenesis in an organ culture system.

Achieving mammalian spermatogenesis in vitro has a long history of research but remains elusive. The organ culture method has advantages over the cell culture method, because germ cells are in situ albeit the tissue as a whole is in vitro. The method was used in the 1960s and 1970s but encountered difficulties in inducing complete meiosis, i.e., in getting meiosis to proceed beyond the pachytene stage. In the present study, we reevaluated the organ culture method using two lines of transgenic mice, Acr-GFP and Gsg2 (haspin)-GFP mice, whose germ cells express green fluorescent protein (GFP) at the mid and end stages of meiosis onward, respectively. Immature testicular tissues from these mice, ranging from 4.5 to 14.5 days postpartum, were cultured on the surface of the medium, providing a liquid-gas interface. Culturing testicular tissues of all ages tested resulted in the expression of both Acr- and Gsg2-GFP. Round spermatids were identified by a combination of Gsg2-GFP expression, cell size, and the presence of a single nucleus with a dot stained by Hoechst. In addition, the chromosome number of one of such presumptive spermatids was found to be 20 by the premature chromosome condensation method. As our semiquantitative assay system using GFP expression grading was useful for monitoring the effects of different environmental factors, including temperature, oxygen concentration, and antiretinoic molecules, further improvement of the culture conditions should be possible in the future.

Sex-discordant twins despite single embryo transfer: a report of two cases.

We report two extremely rare cases in which the patients delivered male and female infants that were dizygotic twins (DZT) despite single embryo transfer. Case 1: The patient was a 35-year-old woman with a 9-year history of unexplained infertility. In an oocyte retrieval cycle, one blastocyst was transferred; at 26 weeks of gestation, she delivered a 704-g female infant and a 420-g male infant by cesarean section. Because both infants were of extremely low birth weight, they were placed in the neonatal intensive care unit. Congenital anomalies were not found in either infant. Case 2: The patient was a 30-year-old woman with a 1-year history of infertility. Hysterosalpingogram revealed bilateral tubal occlusion. In a frozen/thawed cycle one blastocyst was transferred during her natural ovulation cycle. She achieved a pregnancy and delivered a 2,877-g female infant and a 2,544-g male infant at 36 weeks of gestation by cesarean section. The female infant was diagnosed with a neural tube defect. No congenital anomalies were detected in the male infant. We hypothesize that the DZTs might have been the result of concurrent embryo transfer and natural ovulation and intercourse.

A birth of twins-one boy and one girl-from a single embryo transfer and a possible natural pregnancy.

PURPOSE: To describe a rare case of a birth of dizygotic twins with different-sex infants from a single embryo transfer. METHODS AND RESULTS: A patient, who had her right ovary and tube removed, and her husband were treated with ICSI and a single embryo transfer. When a single fresh embryo was transferred on day 4, following oocyte retrieval using GnRH agonist-long protocol, two gestational sacs were recognized at 8 weeks of gestation. Healthy twins with a boy and a girl were delivered at 37 weeks 0 days of gestation by a cesarean section. The boy's weight was 2096g, and his height was 45.0 cm, while the girl's weight was 1988g, and her height was 41.5 cm. Peripheral lymphocyte chromosome analysis of the two infants showed normal karyotype, 46, XY (boy) and 46, XX (girl). CONCLUSIONS: A single embryo transfer could produce different-sex twins.