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PURPOSE: To elucidate the effect of X-ray exposure during hysterosalpingography (HSG) on subsequent laboratory outcomes in in vitro fertilization (IVF). METHODS: A total of 1458 oocytes, consisting of 990 oocytes retrieved from 70 women (89 cycles) who underwent HSG prior to IVF and 468 oocytes from 45 women (57 cycles) who underwent IVF without HSG, were evaluated for their retrieval number, maturity, fertilization, and development post fertilization. X-ray exposure during HSG was recorded as reference air kerma (RAK) (mGy). Subjects were stratified according to the amount of RAK (Nil: IVF without HSG, L-RAK: RAK

PURPOSE: To construct a new embryonic quality scoring system to compare groups of embryos at different developmental stages. METHODS: Based on a hypothesis that the implantation potential of any embryo in an ovum pickup (OPU) cycle remains the same at any stage of development, be it day 2, 3, or 5, a new embryo quality scoring (EQS) system was designed. It was based on the analysis of the clinical results of 1610 single embryo transfers. We validated this scoring system in the comparison of embryonic quality between groups by evaluating the mean scores calculated at day 2, day 3, and day 5 for 957 embryos (150 cycles) from 3 different groups. We then compared EQSs of patients with pregnancy favorable factors (group A) such as young age and high AMH levels, with the patients with contra features (group B). RESULTS: We confirmed that each mean EQS assessed at different stages of embryonic development within the same group was similar. The mean EQSs on day 3 and day 5 in group A were significantly higher than the mean EQSs on days 2, 3, and 5 in group B. CONCLUSION: The novel EQS system proposed by us enables embryonic quality comparison between groups of embryos at different developmental stages.

To investigate the effect of climatic parameters in the mesothermal climate area on clinical pregnancy and live birth following fresh single blastocyst transfer.This study investigated clinical pregnancies and live births that resulted from 555 ovarian stimulation cycles followed by fresh single blastocyst transfer. The samples were stratified according to climatic conditions (low T, temperature 12.9 °C; middle T, 12.9 °C ≤ temperature 22.6 °C; high T, temperature ≥ 22.6 °C; low H, relative humidity 62.1%; middle H, 62.1% ≤ relative humidity 66.5%; high H, relative humidity ≥ 66.5%; short S, sunlight duration 5.2 h; middle S, 5.2 h ≤ sunlight duration 6.7 h; long S, sunlight duration ≥ 6.7 h). Clinical pregnancy and live birth rates among three groups from each climatic parameter were compared. Multivariable analyses were performed to investigate the effects of climatic conditions on blastocyst development, endometrial thickness, clinical pregnancy, and live birth.A statistically significant difference was found in pregnancy rates among low T (48.8%), middle T (37.3%), and high T (36.6%) groups. Multivariable analyses revealed that temperature was associated with clinical pregnancy and live birth rates with adjustment for patient age, BMI, type of ovarian stimulation, endometrial thickness, and expansion grade of the transferred blastocyst. The association between climatic parameters and blastocyst development and endometrial thickness was not confirmed.This study suggests that lower temperatures in the mesothermal climate area could favorably affect the rates of clinical pregnancy and live birth achieved by fresh single blastocyst transfer.

Additional file 2 Fig. S1. Sequence heterogeneities of the 27F primer-annealing site in 16S rRNA genes. Fig. S2. Evaluation of 16S rRNA PCR primers for identification of bacterial species. Fig. S3. Classification results of a mock community analyzed by the different bioinformatics workflows. Fig. S4. Effect of read number on taxonomic classification. Fig. S5. Species composition of a mock community analyzed by FASTQ 16S workflow. Fig. S6. Rarefaction curves of observed OTUs in V3-V4 16S rRNA gene amplicon sequencing of human fecal samples using the MiSeq™ platform. Fig. S7. Species composition of Blautia in human fecal samples. Fig. S8. Species composition of Bacteroides in human fecal samples. Fig. S9. Deviations in the relative abundances of Bifidobacterium species in human fecal samples. Fig. S10. Comparison of species composition of fecal Bifidobacterium between classification methods.

Additional file 7. Alignment search results for V3-V4 amplicon sequencing of the mock community.

Additional file 6. Alignment search results for V1-V9 amplicon sequencing of the mock community.

Background Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. Results We modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. Conclusions Our present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.

Age-dependent decline of mitochondrial function has been proposed to be a main cause of decline of embryo quality. Then, l-carnitine plays important roles in reducing the membranous toxicity of free-fatty acids by forming acyl-carnitine and promoting β-oxidation, preventing cell damage. Recent research revealed that l-carnitine played important roles in vitro in oocyte growth, oocyte maturation and embryo development. However, such beneficial effects of l-carnitine in vivo have yet to be verified. The effect of oral l-carnitine supplementation on embryo quality and implantation potential was examined. A total of 214 patients were included in this study. They all previously received in vitro fertilization-embryo transfer (IVF-ET) and failed to conceive. Then they were administered l-carnitine for 82 days on average and underwent IVF-ET again. There were no significant differences in the total number of retrieved oocytes, and their maturation and fertilization rates between before and after l-carnitine administration. The quality of embryos on Days 3 and 5 after insemination was improved following l-carnitine administration (p

STUDY QUESTION Can preimplantation genetic testing for aneuploidy (PGT-A) improve the live birth rate and reduce the miscarriage rate in patients with recurrent pregnancy loss (RPL) caused by an abnormal embryonic karyotype and recurrent implantation failure (RIF)? SUMMARY ANSWER PGT-A could not improve the live births per patient nor reduce the rate of miscarriage, in both groups. WHAT IS KNOWN ALREADY PGT-A use has steadily increased worldwide. However, only a few limited studies have shown that it improves the live birth rate in selected populations in that the prognosis has been good. Such studies have excluded patients with RPL and RIF. In addition, several studies have failed to demonstrate any benefit at all. PGT-A was reported to be without advantage in patients with unexplained RPL whose embryonic karyotype had not been analysed. The efficacy of PGT-A should be examined by focusing on patients whose previous products of conception (POC) have been aneuploid, because the frequencies of abnormal and normal embryonic karyotypes have been reported as 40–50% and 5–25% in patients with RPL, respectively. STUDY DESIGN, SIZE, DURATION A multi-centre, prospective pilot study was conducted from January 2017 to June 2018. A total of 171 patients were recruited for the study: an RPL group, including 41 and 38 patients treated respectively with and without PGT-A, and an RIF group, including 42 and 50 patients treated respectively with and without PGT-A. At least 10 women in each age group (35–36, 37–38, 39–40 or 41–42 years) were selected for PGT-A groups. PARTICIPANTS/MATERIALS, SETTING, METHODS All patients and controls had received IVF-ET for infertility. Patients in the RPL group had had two or more miscarriages, and at least one case of aneuploidy had been ascertained through prior POC testing. No pregnancies had occurred in the RIF group, even after at least three embryo transfers. Trophectoderm biopsy and array comparative genomic hybridisation (aCGH) were used for PGT-A. The live birth rate of PGT-A and non-PGT-A patients was compared after the development of blastocysts from up to two oocyte retrievals and a single blastocyst transfer. The miscarriage rate and the frequency of euploidy, trisomy and monosomy in the blastocysts were noted. MAIN RESULT AND THE ROLE OF CHANCE There were no significant differences in the live birth rates per patient given or not given PGT-A: 26.8 versus 21.1% in the RPL group and 35.7 versus 26.0% in the RIF group, respectively. There were also no differences in the miscarriage rates per clinical pregnancies given or not given PGT-A: 14.3 versus 20.0% in the RPL group and 11.8 versus 0% in the RIF group, respectively. However, PGT-A improved the live birth rate per embryo transfer procedure in both the RPL (52.4 vs 21.6%, adjusted OR 3.89; 95% CI 1.16–13.1) and RIF groups (62.5 vs 31.7%, adjusted OR 3.75; 95% CI 1.28–10.95). Additionally, PGT-A was shown to reduce biochemical pregnancy loss per biochemical pregnancy: 12.5 and 45.0%, adjusted OR 0.14; 95% CI 0.02–0.85 in the RPL group and 10.5 and 40.9%, adjusted OR 0.17; 95% CI 0.03–0.92 in the RIF group. There was no difference in the distribution of genetic abnormalities between RPL and RIF patients, although double trisomy tended to be more frequent in RPL patients. LIMITATIONS, REASONS FOR CAUTION The sample size was too small to find any significant advantage for improving the live birth rate and reducing the clinical miscarriage rate per patient. Further study is necessary. WIDER IMPLICATION OF THE FINDINGS A large portion of pregnancy losses in the RPL group might be due to aneuploidy, since PGT-A reduced the overall incidence of pregnancy loss in these patients. Although PGT-A did not improve the live birth rate per patient, it did have the advantage of reducing the number of embryo transfers required to achieve a similar number live births compared with those not undergoing PGT-A. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Japan Society of Obstetrics and Gynecology and grants from the Japanese Ministry of Education, Science, and Technology. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A

Recurrent pregnancy loss (RPL) affects 2–5% of all couples. RPL brings not only a grief for the patient but also physical damage on female reproductive organs. Furthermore, no matter how early in the pregnancy is, the loss of a baby is almost the same as the loss of a child. Therefore, a significant role for evaluation after just two losses in patients with no prior live births is strongly recommended. There are a small number of etiologies such as genetic, anatomic, immunologic, endocrine, and others, generally accepted for RPL. On the other hand, it has been widely accepted that aneuploidy is the most common cause of miscarriage. Although each etiologic factor has its treatment strategies, any treatment cannot prevail the aneuploidy. Preimplantation genetic screening (PGS) has been proposed as a method for reducing miscarriage by selecting euploid embryos for transfer. Previous PGS study by FISH failed to demonstrate its validity. However, PGS using recent technologies such as CCS has been showing its validity in preventing miscarriages. PGS with recent technology is feasible to reduce subsequent miscarriages in RPL patients. The drawback factor is that patients have to have ART toward the next conception.

Oocyte in vitro maturation (IVM) has been evoked by Edwards [1] as an elegant proposition for the majority of in vitro fertilization procedures. The goal of clinical application of IVM is to eliminate or significantly reduce important drawbacks of controlled ovarian stimulation, such as drug costs, burden on patients, and the risk of ovarian hyperstimulation syndrome (OHSS), especially in patients with polycystic ovary syndrome (PCOS).

Morphological assessment of human blastocysts has been effective for selecting embryos with high potential. However, they often show repeated shrinkage and expansion toward their hatching. Here we assessed whether capturing morphological changes over time of vitrified-warmed blastocysts could lead to a better selection of viable embryos from shrunken blastocysts.The implantation rates of vitrified-warmed blastocysts that were shrunken or expanded (developing) at the time of loading for transfer were compared among 2,729 cycles that were subjected to single blastocyst transfer. Vitrified (107) and fresh blastocysts (17) were donated for the experimental study. To assess the relationship between morphology (expanded vs. shrunken) and the mitochondrial respiration of blastocysts, the oxygen consumption rate (OCR) was analyzed for 55 specimens using an uncoupler of oxidative phosphorylation. The remaining 69 blastocysts were used for recording morphological changes every 15 min for 48 h after warming.Because there were no surplus embryos, 7 % of the vitrified-warmed blastocysts were shrunken and transferred. The shrunken embryos had sufficient implantation ability (40 %). The OCR of the shrunken embryos was significantly lower than that of their expanded counterparts. Upon exposure to the uncoupler, the OCR of some shrunken embryos increased to levels similar to the expanded specimens. Time-lapse images revealed some shrunken embryos which formed blastocoel by 5 h following warming exhibited developmental competence to the hatched stage.Data of the present study suggest a group of shrunken blastocysts contains many viable and clinically available embryos and time-lapse observation of vitrified-warmed blastocysts is a potential method to distinguish viable embryos from shrunken blastocysts.

Aim Outside of Japan, recombinant-human chorionic gonadotropin (r-hCG) is widely used for the induction of final follicular maturation and early luteinization in women undergoing ovulation induction; whereas in Japan, urine-derived hCG (u-hCG) is predominantly used. The primary objective of this study was to demonstrate the non-inferiority of r-hCG to u-hCG for ovulation induction, as assessed by the ovulation rate. Methods This was an open-label, parallel-group, randomized, multicenter, phase III trial in Japanese women with anovulation or oligo-ovulation secondary to hypothalamic–pituitary dysfunction or polycystic ovary syndrome, undergoing ovulation induction with recombinant-human follicle-stimulating hormone. The women were randomized (2:1) to receive either a single 250 μg s.c. dose of r-hCG or a single 5000 IU i.m. dose of u-hCG for ovulation triggering. Results Eighty-one women were randomized to either r-hCG (n=54) or u-hCG (n=27). Ovulation occurred in 100% of the participants and treatment with r-hCG was observed to be non-inferior to u-hCG for ovulation induction. Overall, the type and severity of adverse events were as expected for women receiving fertility treatment. Conclusion This study demonstrated that r-hCG was non-inferior to u-hCG for inducing ovulation. Furthermore, r-hCG demonstrated an expected safety profile, with no new safety concerns identified.

Infertility specialists have been struggling to deal with poor responders, reported to range from 9 to 24 %. However, the incidence is abruptly increasing lately due to the increasing population of aged infertile women. The long gonadotropin-releasing hormone (GnRH) protocol is the first line of treatment if more than plural numbers of oocytes are expected, which is followed by the short protocol, and the GnRH antagonist stimulation is used as the last treatment option. Among the GnRH agonist protocols, the microdose agonist flare with oral contraceptive (OC) pretreatment appears more effective. Natural cycle or mild stimulation is used when the stimulation protocols fail. IVM is an alternative choice when none of the above-mentioned protocols succeed. Growth hormone (GH) appears to have a beneficial effect, but indications for using GH as well as growth hormone-releasing hormone (GH-RH) are not well defined. In addition, it is too early to determine if androgen pretreatment [dehydrepiandrosterone sulfate (DHEA), testosterone] is beneficial in the treatment of poor responders. It is most important for ART physicians to evaluate the patient response before starting the stimulation and choose the best protocol for the poor responder.

In this study, to avoid the virus contamination in the maturation medium by addition of donor follicular fluid (FF) from other in vitro fertilization (IVF) cycles, we examined the outcome of in vitro maturation (IVM) protocol using medium containing patient's own serum. Data were retrospectively collected from 68 irregularly cycling women (31 ± 3.6 years) who were diagnosed as having polycystic ovarian syndrome. Immature oocytes were retrieved transvaginally 36 h after an injection of 10,000 IU hCG. Immature oocytes were matured for 24–26 h in medium supplemented with 10% (v/v) heat-inactivated patient's own serum (group S) or with 20% (v/v) heat-inactivated FF from donors who had undergone IVF cycles (group FF). All mature oocytes were inseminated by intracytoplasmic sperm injection. There were no statistical differences in the rates of maturation (49.2% vs. 45.6%), fertilization (85.0% vs. 84.2%), and pregnancy (16.0% vs. 15.4%) between group S and group FF. The supplementation of patient's own...

L-Glutamine has been shown to play an important role during in vitro culture of mammalian embryos. However, it is easily decomposed into ammonium, which is believed to have deleterious effects on preimplantation embryos. In this study, we assessed prospectively the developmental competence of human embryos cultured in medium containing L-glutamine or a novel stable glutamine derivative and vitamins. The subjects of this study were 41 women who underwent IVF/ET treatment from September to November 2006 and from whom 6 or more oocytes were retrieved. Sibling oocytes were randomly divided into EA/BA (EmbryoAssyst and BlastAssyst containing a novel stable glutamine derivative and vitamins), and BAS groups (BlastAssyst system containing L-glutamine). There was no difference in pronuclear formation rate between EA/BA and BAS (74 vs. 69%). The blastulation rates of embryos based on the number of zygotes cultured in EA/BA on Days 5 (Day 0=insemination, 54%) and 6 (63%) were significantly higher (P

The aim of this study was to establish a culture system to improve the meiotic competence of porcine oocyte-granulosa cell complexes (OGCs) obtained from preantral or early antral follicles. Porcine OGCs were recovered from follicles with diameters of 230-300 (preantral follicles), 300-500, and 500-700 mum (early antral follicles) using scalpels. The OGCs were cultured for 2 weeks in culture medium. We examined the effects of the sizes of the follicles from which OGCs were recovered, the concentrations of polyvinylpyrrolidone (PVP, 0-8%) in the culture medium, and 2 types of culture dish (Falcon 3002 vs 1007) on formation of the antrum of OGCs. After culture, the oocytes were matured for 44 h to assess their meiotic competence. OGCs recovered from small follicles (230-500 microm) required longer (P

In-vitro maturation (IVM) of immature oocytes has been proposed as a potential alternative to conventional IVF treatment following ovarian stimulation. However, the effects of the oocyte retrieval conditions on subsequent development have not been well understood. This study assessed the effects of different aspiration vacuums during oocyte retrieval on the developmental competence of immature oocytes following IVM, IVF and embryo transfer, retrospectively. Immature oocytes were aspirated with 20-gauge needles with a vacuum of 180 or 300 mmHg. Immature oocytes were cultured in IVM medium for 26 h. All mature oocytes were inseminated by intracytoplasmic sperm injection (ICSI). Embryo transfer was carried out 2 or 3 days after ICSI. The percentage of cumulus-cell enclosed oocytes and of transferable embryos per retrieved oocytes in 180 mmHg (69.7% and 23.8%, respectively) were significantly higher (P < 0.01) than those in 300 mmHg (46.2% and 12.8%, respectively). The ongoing pregnancy rate per retrieval cycle in 180 mmHg (30%) was higher (P < 0.01) than that in 300 mmHg (4.3%). The data indicate that lower pressure of vacuum aspiration with a 20-gauge needle improves the developmental competence of immature oocytes following IVM, IVF and embryo transfer.

Successful pregnancies from IVF of immature oocytes in unstimulated cycles (IVM-IVF) have been obtained since 1999 at our clinic. However, pregnancy rate of IVM-IVF remains marginal. The objective of the present study was to investigate whether priming patients with HCG would improve its outcome. Forty-two cycles were primed with HCG and 119 used as control were not primed. Those patients were classified into three groups with regular cycles, irregular cycles or PCOS. The number of oocytes retrieved and oocyte maturation rate at 24 h increased significantly with HCG compared to control in the patients with either regular or irregular cycles, but not in PCOS. There were no significant differences in fertilization rate by ICSI, embryonic development and pregnancy rate in any groups. The present study suggests that HCG priming increases the number of oocytes retrieved in some patient groups, but dose not improve clinical outcome.

Introduction: Multiple trials of immunotherapy for reproductive failure have failed to demonstrate significant benefit. A reason for this may be the lack of a test that selects which women will benefit from treatment. High density of uterine naturak killer (uNK) cells has been associated with recurrent miscarriage (RM) and prednisolone was shown to reduce this uNK cell density. Hence we carried out a double blind randomised placebo controlled trial of prednisolone versus placebo in early pregnancy for women with RM and increased uNK cell density. The aim of this pilot trial was to assess the feasibility of recruitment, integrity of trial procedures and potential efficacy for future power calculations.

Objective: Determine if aspirin inhibits the IL-1β–stimulated expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and prostaglandin E 2 (PGE 2 ) in rat ovarian dispersates cultured in vitro . Design: Prospective, controlled in vitro study. Setting: Academic research laboratory. Animals: Ovaries collected from immature rats. Intervention(s): Ovaries were collected from immature rats and enzymatically dispersed. Ovarian dispersates were placed into plates containing media alone or media supplemented with IL-1β (100 U/mL) and varying concentrations of aspirin (0, 1, 3, 5 and 10 mM). Ovarian dispersates were cultured in a humidified environment of 5% CO 2 in air at 37°C for 24 or 48 hours. Main Outcome Measure(s): Twenty-four– and 48-hour iNOS, nitrite (a stable metabolite of NO), and PGE 2 levels were determined from ovarian dispersates cultured in vitro. Result(s): Administration of IL-1β increased nitrite and PGE 2 levels over that observed in the control group after culture of ovarian dispersates for 24 and 48 hours. Aspirin dose dependently reduced the IL-1β–stimulated increase in nitrite production from ovarian dispersates after culture for 24 and 48 hours. Aspirin completely (24 hours) or dose dependently (48 hours) prevented the IL-β–stimulated increase in PGE 2. Coadministration of IL-1β and aspirin (10 mM) attenuates IL-1β–stimulated iNOS expression after culture for 24 and 48 hours. Conclusion(s): Aspirin significantly inhibits the IL-1β–stimulated expression of iNOS, NO, and PGE 2 in ovarian dispersates cultured in vitro.

We report here the first case of successful pregnancy and delivery after the blastocyst transfer of twice-vitrified embryos produced following in vitro maturation (IVM) and ICSI. The patient received 5000 IU hCG on day 12 of the treatment cycle, and oocyte retrieval was carried out 36 h after hCG injection. A total of 22 immature oocytes were obtained. Following incubation for 26 h in IVM medium, 15 oocytes (68.2%) reached metaphase II stage. In total, 13 oocytes (86.7%) were fertilized after ICSI with the husband's sperm, and 11 embryos at the pronuclear stage and two cleaved embryos on day 2 were vitrified because of thin endometrial thickness. Eight cryopreserved embryos at the pronuclear stage were warmed and cultured until the day 3 stage. Three embryos were transferred, and three embryos were twice vitrified. Unfortunately, these transferred embryos did not implant. Three twice-vitrified embryos were rewarmed and cultured until the day 5 stage, and two embryos were transferred. The second transfer attempt of twice-vitrified embryos resulted in the full-term delivery of a healthy infant. This case report demonstrates that twice-vitrified embryos, developed using an IVM protocol, retain the developmental competence for full-term, healthy infants.

Functional and structural changes in the mammalian ovary are coordinately regulated by the pituitary glycoprotein hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), leading to follicular development, ovulation and transformation of follicles into corpus lutea. To investigate protein profiles during these processes of the mouse ovarian cycle, we applied combined methods (two-dimensional gel electrophoresis [2-DE] for separation and visualization of proteins plus matrix laser desorption/ionization time-of-flight mass spectrometry [MALDI-TOF/MS] analysis for protein identification) for comparative proteomic analysis using immature mice at 3 weeks of age. Protein profiles were obtained from proteins extracted from intact ovaries that had been collected from pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed immature mice at 0 (no PMSG), 24 and 48 h post PMSG, as well as at 10 and 20 h post hCG. The results showed that 1028 common protein spots were found in representative gels that had been separated in the 3 to 11 pH range and the 15-200 kDa range, 253 protein spots (24.6%) of which were differentially expressed (p

The expression of class II major histocompatibility complex (MHC) antigen on the membrane of mouse sperm head was clearly demonstrated by indirect immunofluorescence (IIF) test, enzyme immunoassay (EIA), and mixed lymphocyte sperm reaction (MLSR) as well as the examination of its expression at transcriptional levels by nothern dot blotting. Furthermore, the roles of class II MHC antigen of sperm in fertilization were investigated with an in vitro fertilization (IVF) system. The successful ratio of IVF was significantly decreased by treatment of sperm with anticross-reactive region of class II MHC antigen monoclonal antibody (MAb) but not with antiprivate region MAb. These results were not due to disturbances of sperm mobility by these MAbs. It was strongly suggested that the monomorphic or its related region of class II MHC antigen on sperm played an important role in the recognition between sperm and egg in fertilization.

BACKGROUND: Obtaining mature human follicles from cultured ovarian tissue may be beneficial for clinical use for women who wish to preserve fertile competence. However, the methodology of culture such as culture condition and gas atmosphere has not been well established in humans. Therefore, we investigated the effect of oxygen concentration in organ culture in order to establish an ovarian tissue culture method. METHODS: Ovarian tissue was obtained from 26-35-year-old women undergoing removal of a benign tumor (n = 12) or caesarean section (n = 16). The ovarian cortical tissues were cultured on a cell culture insert for 15 days under high (100%) and low (air, 20%) oxygen concentrations and then inspected for follicle development with light and electron microscopy. Estradiol and progesterone concentrations in the medium during culture were measured. RESULTS: The ultrastructure and the function of hormone secretion in the cultured tissues were well preserved after organ culture. The follicles developing under high oxygen were larger and more matured than those developing under low oxygen (P < 0.05). CONCLUSIONS: Human ovarian tissues can be cultured for 15 days under high oxygen concentration with the organ culture system used here. This technique could make it possible to utilize ovarian tissue for preservation of reproductive competence in cancer patients.

We previously identified a novel gonad-specific expression gene (Gse) and investigated its expression during gametogenesis in the mouse testis and ovary. In this study, we generated a polyclonal antibody to GSE protein and determined the profiles of the protein's expression in germ cells and preimplantation embryos in detail using immunocytochemical and immunofluorescence staining. In a Western blot analysis, the anti-GSE antibody recognized long and short isoforms (approximately 27.6 kDa and 23.1 kDa) of the protein in the mouse testis and the long isoform in the ovary. In the mouse testis, GSE protein was expressed in spermatocytes I in the pachytene stage, round spermatids, and elongated spermatids. In the mouse ovary, the protein was located in the cytoplasm and nucleus of all oocytes regardless of the stage of the ovarian follicles. In preimplantation embryos from the pronuclear to blastocyst stage, however, GSE protein was mainly detected in the nuclei of cells. At the blastocyst stage, the protein was confirmed to have accumulated in the inner cell mass (ICM), whereas it had mostly disappeared from the trophectoderm (TE). These findings suggest that GSE protein may play a role in the establishment of nuclear totipotency and may be associated with early lineage specification.

The developmental rate of a blastocyst is considered one of the main estimates for evaluating the implantation potential of embryos. Day 6 blastocysts have been reported to be much less viable than day 5 blastocysts. Regarding implantation, the implantation window is advanced due to a background of high sex hormones, and slower growing embryos may not implant because of possible desynchrony with the implantation window. The aim of this study was to investigate the efficacy of cryopreservation of such embryos and subsequent synchronization of embryo transfer with endometrial status. The results of 122 day 6 blastocysts transferred in the clinic were retrospectively examined. Pregnancy rates were compared between the stimulation cycle and hormone replacement cycle in terms of the method of endometrial preparation. Fifty-five day 6 blastocysts were transferred onto the stimulation cycle endometrium in 37 women, resulting in a 5.5% viable pregnancy rate. On the other hand, 67 day 6 blastocysts were transferred onto endometrium prepared by exogenous hormones in 40 women, resulting in a 26.9% viable pregnancy rate (P < 0.01). Consequently, the difference was highly significant. In conclusion, synchronous transfer of slow-growing embryos using the freeze-thaw technique contributes to a positive outcome.

In this study, luteinized human granulosa cells (GC) obtained during in vitro fertilization procedures were used as a model system to evaluate the effects of ethanol (EtOH), a well-known reproductive toxin, on epidermal growth factor (EGF) and gonadotropin-stimulated steroidogenesis. Our results demonstrate that the basal progesterone (P4) and estradiol (E2) secretion by human GC in vitro was dependent on the ovarian stimulation protocol. EGF significantly enhanced P4, but not E2, secretion in human GC from clomiphene citrate (CC), human menopausal gonadotropin (hMG), and hMG/gonadotropin-releasing hormone agonist (GnRH-a)-treated patients. The effects of EGF plus luteinizing hormone (LH) were additive in cells from the CC group, but less than additive in hMG and hMG/GnRH-a groups. EtOH at 20 mM or more inhibited EGF stimulated P4 secretion in human GC from all three patient groups. EtOH inhibited P4 secretion stimulated by EGF and LH cotreatment in the CC and hMG/GnRH-a groups, but not in human GC from the hMG-treated patients. These results suggest that basal and EGF or LH-stimulated P4 secretion by human GC, as well as the effects of EtOH, are profoundly influenced by the follicle's hormonal milieu.

Our purpose was to investigate the influence of in vitro oocyte aging on fertilization and subsequent embryonic development following subzonal sperm injection with reference to spontaneous zona hardening in the mouse. First cleavage rate was significantly higher in long vs short (69 vs 41%, P < 0.001). However, significantly higher blastocyst formation and hatching were observed in short than long (50 vs 7%, P < 0.001, and 86 vs 50%, P < 0.001, respectively). No significant differences were found for two pronuclei formation between short and long (30 vs 27%). Sham injection revealed a significantly higher parthenogenetic activation in long than short (19 vs 2%, P < 0.001). Zona digestion required a significantly longer time for long compared to short (trypsin—37 vs 29 min, P < 0.01; pronase—17 vs 14 min, P < 0.001). Prolonged culture of mature mouse oocytes in vitro alters the zona pellucida, increases the rate of parthenogenetic activation by subzonal sperm injection, and impairs subsequent embryonic development. Zona hardening appears to be an indicator of oocyte aging.