Comparative Analysis of Two NGS-Based Platforms for Product-of-Conception Karyotyping.

Cytogenetic information about the product of conception (POC) is important to determine the presence of recurrent chromosomal abnormalities that are an indication for preimplantation genetic testing for aneuploidy or structural rearrangements. Although microscopic examination by G-staining has long been used for such an evaluation, detection failures are relatively common with this method, due to cell-culture-related issues. The utility of low-coverage whole-genome sequencing (lcWGS) using short-read next-generation sequencing (NGS) has been highlighted recently as an alternative cytogenomic approach for POC analysis. We, here, performed comparative analysis of two NGS-based protocols for this purpose based on different short-read sequencers (the Illumina VeriSeq system using a MiSeq sequencer and the Thermo Fisher ReproSeq system using an Ion S5 sequencer). The cytogenomic diagnosis obtained with each NGS method was equivalent in each of 20 POC samples analyzed. Notably, X chromosome sequence reads were reduced in some female samples with both systems. The possibility of low-level mosaicism for monosomy X as an explanation for this was excluded by FISH analysis. Additional data from samples with various degrees of X chromosome aneuploidy suggested that it was a technical artifact related to X chromosome inactivation. Indeed, subsequent nanopore sequencing indicated that the DNA in the samples showing the artifact was predominantly unmethylated. Our current findings indicate that although X chromosome data must be interpreted with caution, both the systems we tested for NGS-based lcWGS are useful alternatives for the karyotyping of POC samples.

Supplementation with autologous adipose stem cell-derived mitochondria can be a safe and promising strategy for improving oocyte quality.

PURPOSE: In our previous study, we confirmed that the supplementation of vitrified-warmed murine oocytes with autologous adipose stem cell (ASC)-derived mitochondria during intracytoplasmic sperm injection enhances post-fertilization developmental competence in mice. To ensure the safety of this technology, we conducted a thorough study in mice to investigate the potential presence of specific malformations in offspring developed from this approach. METHODS: A transgenerational comparative analysis was conducted on founder mice from embryos that developed after mitochondrial supplementation, and two subsequent generations. Reproductive performance, body growth rate, histopathological parameters, hematological parameters, daily activity patterns, and daily body temperature changes in male and female mice across these three generations were assessed in comparison to wild-type mice of the same age. RESULTS: Both male and female animals in all three generations showed comparable reproductive performance to the control group. Additionally, body growth rate by the age of 8 weeks were found to be comparable to controls across all three generations. Notably, no significant histopathological abnormalities were detected in vital organs, including the brain, heart, liver, kidneys, lungs, ovaries, and testes, in any individuals from the studied cohorts. The blood parameters were consistent with the control data. The continuous monitoring of activity and body temperature changes (both day and night) over a 1-week period revealed a pattern closely resembling that observed in the control animals. CONCLUSION: Injection of ASC-mitochondria into oocytes may be a promising technique to support developmental potential without causing adverse epigenetic events in the offspring in mice. However, before considering clinical application, additional safety screening using larger animals or non-human primates is essential.

Lifestyle and fertility-specific quality of life affect reproductive outcomes in couples undergoing in vitro fertilization.

Objective: A Mediterranean dietary pattern, sleeping habits, physical activity, and lifestyle appear to affect reproductive health. There are few reports about whether fertility-specific quality of life (QOL) is linked to infertility treatment outcomes. The aim of this study is to investigate when lifestyle factors and fertility-specific QOL are comprehensively considered, which factors influence assisted reproductive technology (ART) outcomes. Methods: This prospective cohort includes 291 women undergoing a first ART treatment at multiple centers in Japan and was designed to evaluate the influence of diet, physical activity, sleeping pattern, computer use duration, and fertility-specific quality of life tool (FertiQoL) score on ART treatment outcomes using a questionnaire. The primary endpoint was the good-quality blastocyst rate per oocyte retrieval and the secondary endpoints were a positive pregnancy test and gestational sac (GS) detection. Results: The good-quality blastocyst rate per oocyte retrieval tended to be negatively associated with frequent fish consumption. After all embryo transfer (ET) cycles, a positive pregnancy test tended to be positively associated with longer sleep and longer computer use (OR = 1.6, 95% CI = 0.9-2.7 and OR = 1.7, CI = 1.0-2.8, respectively) and negatively associated with a smoking partner (OR = 0.6, CI = 0.3-1.0). GS detection was positively and significantly associated with frequent olive oil intake and longer computer use (OR = 1.7, CI = 1.0-3.0 and OR = 1.7, CI = 1.0-3.0, respectively). After ET cycles with a single blastocyst, a positive pregnancy test was positively and significantly associated with longer computer use (OR = 2.0, CI = 1.1-3.7), while GS detection was significantly more likely in women with longer computer use (OR = 2.1, CI = 1.1-3.8) and tended to be more likely in women with a higher FertiQoL Total scaled treatment score (OR = 1.8, CI = 1.0-3.3). p < 0.05 was considered statistically significant and 0.05 ≤ p <0.01 as tendency. Conclusions: Olive oil may be an important factor in dietary habits. Fertility-specific QOL and smoking cessation guidance for partners are important for infertile couples.

Impact of Lactobacillus in the uterine microbiota on in vitro fertilization outcomes.

Abundant intrauterine Lactobacillus is associated with good in vitro fertilization (IVF) outcomes; however, whether specific species of Lactobacillus have any benefit remains unclear. So we examine the effect of Lactobacillus on the clinical outcomes of IVF at the species level. Uterine microbiota were classified as either Lactobacillus-dominant (LD) or non-Lactobacillus-dominant. In the LD group, we further investigated the clinical results for each Lactobacillus species and evaluated them in relation to IVF outcomes. In Uterine microbiome analysis, Lactobacillus was the most abundant, with the four species of L. crispatus, L. iners, L. gasseri, and L. jensenii accounting for the great majority. We compared the clinical outcomes of single frozen-thawed embryo transfer conducted by Lactobacillus species and found that the implantation rate was lowest in those in whom L. iners was dominant. This study is the first to conduct a species-level analysis of the uterine microbiota and report on a detailed investigation of Lactobacillus, which was believed to be particularly helpful for pregnancy.

Exploring the Impact of Endometrioma Aspiration and Dienogest Combination Therapy on Cyst Size, Inflammatory Cytokines in Follicular Fluid and Fertility Outcomes.

Endometriomas (chocolate cysts) are cystic lesions that can develop on ovaries, and are characterized by the presence of ectopic endometrial tissue or similar tissue. Such lesions can cause a decline in the number and quality of oocytes, and lead to implantation failure. In this study, we retrospectively assessed the efficacy of repeated endometrioma aspiration and dienogest combination therapy in patients suffering endometriosis-associated infertility with endometriomas. A comparison was made between a treated group that underwent combination therapy followed by controlled ovarian hyperstimulation (COH) (n = 30) and a control group that did not undergo treatment (n = 40), at the IVF Osaka Clinic from September 2019 to September 2021. There were no differences in patient background between the two groups. A reduction in endometrioma size continued for 12 months after treatment. The numbers of follicles that developed to 15 mm or greater in size following COH and mature oocytes were significantly lower in the treated group compared to those in the control group. The levels of inflammatory cytokines in the follicular fluid significantly decreased in the treated group (p < 0.05). In patients in the treated group who underwent a second ova retrieval, the results were compared between those in the first ova retrieval (immediately after the end of treatment) and those in the second ova retrieval (four months after the first retrieval). The numbers of follicles following COH, retrieved, mature and fertilized ova were significantly increased in the second ova retrieval.

Exogenous GSH Supplementation to Raw Semen Alters Sperm Kinematic Parameters in Infertile Patients.

Glutathione is an important antioxidant found in all mammalian cells. Sperm motility is positively correlated with seminal reduced glutathione (GSH) levels, and infertile men are known to have lower GSH levels. Studies on GSH supplementation in improving sperm functions in infertility patients are limited. Here, we re-investigate the effect of exogenous GSH supplementation on human sperm motility and kinematic parameters. Residual semen samples from 71 infertility patients who came for routine semen analysis for infertility assessment were studied. Liquefied raw semen was supplemented with GSH (0-10 mM) for 1 h. The untreated sample was the blank control. Only a 5 mM concentration was tested in all 71 samples. After two washes, the sperm was incubated and then analyzed for sperm motility and kinematic parameters by computer-assisted semen analysis (CASA), followed by adenosine triphosphate (ATP), reactive oxygen species (ROS) levels, free thiols, and DNA damage analyses. At 2 hrs post-treatment, GSH supplementation significantly altered many of the kinematics, compared to the control. Straight line velocity (VSL) (p = 0.0459), curvilinear velocity (VCL) (p < 0.0001), average path velocity (VAP) (p < 0.0001), and lateral head amplitude (ALH) (p < 0.0001) were decreased, whereas straightness (STR) (p = 0.0003), linearity (LIN) (p = 0.0008), and beat cross frequency (BCF) (p = 0.0291) were increased in 5 mM group. Wobble (WOB) (p = 0.4917), motility (MOT) (p = 0.9574), and progressive motility (PROG) (p = 0.5657) were unchanged. ATP level was significantly increased in the 5 mM group (p < 0.05). It is concluded that exogenous GSH supplementation does alter sperm kinematics in humans. These altered kinematic parameters together with increased energy (ATP) may have a positive role in influencing the success rates of ART procedures.

Relation between semen oxidative reduction potential in initial semen examination and IVF outcomes.

Purpose: The MiOXSYS system is a new technique to analyze the semen oxidative reduction potential (ORP) that may use to classify the level of sperm DNA integrity. It does not clearly explain how the semen ORP values could help to change the IVF outcomes. We have analyzed correlations between semen ORP value and the IVF results. Methods: Four hundred and thirty couples were enrolled. The male counterparts were divided into two groups according to their semen ORP values and compared the fertilization rate, cell cleavage rate, and embryo quality, following the IVF procedures. The relations between ORP values and the clinical pregnancy, live birth, and abortion rates were analyzed. Results: The ORP values show negative and positive correlations with some conventional semen parameters. The fertilization and the cleavage rate did not show any differences in those two groups, but the transferable embryo rate was significantly high in patients with high semen ORP. However, the patients with high ORP show a tendency to lower clinical pregnancy with a low abortion rate compared to the low ORP group. Conclusion: The main purpose of measuring the ORP value in semen is still questionable and shows controversial results.

Mitochondrial Transfer into Human Oocytes Improved Embryo Quality and Clinical Outcomes in Recurrent Pregnancy Failure Cases.

One of the most critical issues to be solved in reproductive medicine is the treatment of patients with multiple failures of assisted reproductive treatment caused by low-quality embryos. This study investigated whether mitochondrial transfer to human oocytes improves embryo quality and provides subsequent acceptable clinical results and normality to children born due to the use of this technology. We transferred autologous mitochondria extracted from oogonia stem cells to mature oocytes with sperm at the time of intracytoplasmic sperm injection in 52 patients with recurrent failures (average 5.3 times). We assessed embryo quality using the following three methods: good-quality embryo rates, transferable embryo rates, and a novel embryo-scoring system (embryo quality score; EQS) in 33 patients who meet the preset inclusion criteria for analysis. We also evaluated the clinical outcomes of the in vitro fertilization and development of children born using this technology and compared the mtDNA sequences of the children and their mothers. The good-quality embryo rates, transferable embryo rates, and EQS significantly increased after mitochondrial transfer and resulted in 13 babies born in normal conditions. The mtDNA sequences were almost identical to the respective maternal sequences at the 83 major sites examined. Mitochondrial transfer into human oocytes is an effective clinical option to enhance embryo quality in recurrent in vitro fertilization-failure cases.

Influence of climatic conditions in the mesothermal climate area on pregnancies following elective fresh single blastocyst transfer.

PURPOSE: To investigate the effect of climatic parameters in the mesothermal climate area on clinical pregnancy and live birth following fresh single blastocyst transfer. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Mitochondrial function of human embryo: Decline in their quality with maternal aging.

Background: Female fertility declines with age, due to increased chromosomal aneuploidy and possible reduced mitochondrial function in the embryo. Methods: This review outlines how mitochondrial function in human embryos, as predicted from oxygen consumption rate (OCR) measurements, changes in preimplantation stage, and what factors, particularly maternal age, affect mitochondrial function in embryos. Main findings: The structure of the mitochondrial inner membrane and its respiratory function developed with embryo development, while the copy number of mitochondrial DNA per specimen was transiently reduced compared with that of the oocyte. The undifferentiated state of the inner cell mass cells appears to be associated with a low OCR. In contrast, the copy number of mitochondrial DNA increased in trophoblast cells and mitochondrial aerobic metabolism increased.The OCRs at morulae stage decreased with maternal age, but there was no relationship between maternal age and the copy number of mitochondrial DNA at any stages. The higher oxygen spent at the morula stage; the shorter time was needed for development to the mid-stage blastocyst. Conclusions: The mitochondrial respiratory function of human embryos developed along with embryonic growth. Mitochondrial function at morula stage declined with their maternal age and reduced mitochondrial function decreased the rate of development from morula to blastocyst.

Analysis of clinical outcomes and meiotic segregation modes following preimplantation genetic testing for structural rearrangements using aCGH/NGS in couples with balanced chromosome rearrangement.

Purpose: To retrospectively evaluate the effectiveness of PGT-SR by array comparative genomic hybridization (aCGH) or next-generation sequencing (NGS) in preventing recurrent miscarriages. Methods: Thirty one couples with balanced translocation who underwent 68 PGT-SR cycles between 2012 and 2020 were evaluated. A total of 242 blastocysts were biopsied for aCGH or NGS. The genetically transferable blastocysts were transferred in the subsequent frozen-thawed single embryo transfer cycle. Results: The genetically transferable blastocyst rate was 21.2% (51/241). Thirty five genetically transferable blastocysts were transferred into the uterine cavity. The clinical pregnancy rate was 57.1% (20/35), and the ongoing pregnancy rate was 100.0% (20/20). The incidence of interchromosomal effect (ICE) was influenced by ovarian stimulation protocol, female age, and carrier's gender, but dependent on the types of balanced translocation carriers. Furthermore, there was no significant difference in meiotic segregation modes in ovarian stimulation protocols and carrier's gender. Interestingly, the incidence of adjacent-1 segregation in ≧40 years group increased significantly compared with <35 years group. Conclusions: For the first time in Japan, we show the effectiveness of PGT-SR using aCGH or NGS, which enables comprehensive analysis of chromosomes, in the prevention of recurrent miscarriages. Furthermore, our results may support better genetic counseling of balanced translocation carriers for PGT-SR cycles.

Prevention of intrauterine fetal growth restriction by administrating C1q/TNF-related protein 6, a specific inhibitor of the alternative complement pathway.

PURPOSE: The latest treatments do not sufficiently prevent miscarriage and fetal growth restriction (FGR) in pregnant women. Here, we assessed the effects of a human protein, CTRP6, that specifically inhibits the activation of the alternative complement pathway on miscarriage, fetal and placental development. METHODS: Pregnant CBA/J mice mated with DBA/2 male mice as a model of spontaneous abortion and FGR were randomly divided into the control and CTRP6 groups. In the CTRP6 group, the mice were intravenously administered CTRP6 on days 4.5 and 6.5 post-conception (dpc). The abortion rate and fetal and placental weights on 14.5 dpc were examined. Remodeling of the spiral artery was also assessed. RESULTS: The abortion rate in the CTRP6 group (13%) was reduced compared to the control group (21%), but there was no statistical difference. The placental and fetal weights in the CTRP6 group were also heavier than those in the control (P < 0.05). Moreover, the thickness of the blood vessel wall in the CTRP6 group was significantly thinner than that in the control (P < 0.05) and comparable to that in the non-abortion model (CBA/J x BALB). The ratio of the inner-per-the-outer diameter of the spiral artery increased more in the CTRP6 group than that in the control (P < 0.05). As well, the Th1/Th2 cytokine ratio was significantly reduced by CTRP6 treatment. CONCLUSIONS: Taken together, the supplementation with a protein that regulates the alternative complement pathway in vivo improves FGR and promotes spiral artery remodeling in a mouse model of miscarriage and FGR.

Mitochondria Transfer from Adipose Stem Cells Improves the Developmental Potential of Cryopreserved Oocytes.

Although it is not a well-established technology, oocyte cryopreservation is becoming prevalent in assisted reproductive technologies in response to the growing demands of patients' sociological and pathological conditions. Oocyte cryopreservation can adversely affect the developmental potential of oocytes by causing an increase in intracellular oxidative stresses and damage to the mitochondrial structure. In this study, we studied whether autologous adipose stem cell (ASC) mitochondria supplementation with vitrified and warmed oocytes could restore post-fertilization development that decreased due to mitochondrial damage following cryopreservation. ASC mitochondria showed similar morphology to oocytes' mitochondria and had a higher ATP production capacity. The vitrified-warmed oocytes from juvenile mice were supplemented with ASC mitochondria at the same time as intracellular sperm injection (ICSI), after which we compared their developmental capacity and the mitochondria quality of 2-cell embryos. We found that, compared to their counterpart, mitochondria supplementation significantly improved development from 2-cell embryos to blastocysts (56.8% vs. 38.2%) and ATP production in 2-cell embryos (905.6 & 561.1 pmol), while reactive oxygen species levels were comparable. With these results, we propose that ASC mitochondria supplementation could restore the quality of cryopreserved oocytes and enhance the embryo developmental capacity, signifying another possible approach for mitochondrial transplantation therapy.

Stoichiometric CT number calibration using three-parameter fit model for ion therapy.

PURPOSE: Treatment planning for ion therapy involves the conversion of computed tomography number (CTN) into a stopping-power ratio (SPR) relative to water. The purpose of this study was to create a CTN-to-SPR calibration table using a stoichiometric CTN calibration model with a three-parameter fit model for ion therapy, and to demonstrate its effectiveness by comparing it with a conventional stoichiometric CTN calibration model. METHODS: We inserted eight tissue-equivalent materials into a CTN calibration phantom and used six CT scanners at five radiotherapy institutes to scan the phantom. We compared the theoretical CTN-to-SPR calibration tables created using the three-parameter fit and conventional models to the measured CTN-to-SPR calibration table in three tissue types: lung, adipose/muscle, and cartilage/spongy bone. We validated the estimated SPR differences in all cases and in a worst-case scenario, which revealed the largest estimated SPR difference in lung tissue. RESULTS: For all cases, the means ± standard deviations of the estimated SPR difference for the three-parameter fit method model were -0.1 ± 1.0%, 0.3 ± 0.7%, and 2.4 ± 0.6% for the lung, adipose/muscle, and cartilage/spongy bone, respectively. For the worst-case scenario, the estimated SPR differences of the conventional and the three-parameter fit models were 2.9% and -1.4% for the lung tissue, respectively. CONCLUSIONS: The CTN-to-SPR calibration table of the three-parameter fit model was consistent with that of the measurement and decreased the calibration error for low-density tissues, even for the worst-case scenario.

The novel oral gonadotropin-releasing hormone receptor antagonist relugolix is a new option for controlled ovarian stimulation cycles.

Purpose: Relugolix is an oral gonadotropin-releasing hormone antagonist (GnRHant), which was first introduced in 2019. This study investigated the effects of the conventional injectable GnRHant formulation and this new oral GnRHant formulation on controlled ovarian stimulation (COS) cycles. Methods: Relugolix was administered in 126 cycles and conventional GnRHant injection was administered in 658 cycles (controls). The follicle stimulation was performed by an antagonist method, and for final oocyte maturation, recombinant human chorionic gonadotropin (rHCG), or gonadotropin-releasing hormone agonist (GnRHa), or both (dual trigger) were selected. The number of retrieved oocytes was counted and then they were evaluated for subsequent development up to cleavage stage. Results: The number of retrieved oocytes which was the primary outcome of this research was affected by the combination of GnRHant type and the final oocyte maturation agent. The combination of relugolix and a GnRHa trigger showed a significantly lower number of retrieved oocytes (p < 0.001) than the other combinations. Conclusions: Relugolix is a new option for COS cycles, but should be carefully combined with the final maturation agent. Clinical trial approval: This study was conducted after approval by the Medical Corporation Sankeikai Institutional Ethics Committee (approval number: 2019-34).

Usefulness of combined NGS and QF-PCR analysis for product of conception karyotyping.

Purpose: Since chromosomal abnormalities can be detected in more than half of miscarriages, cytogenetic testing of the product of conception (POC) can provide important information when preparing for a subsequent pregnancy. Conventional karyotyping is the common diagnostic method for a POC but can be problematic due to the need for cell culture. Methods: We here conducted shallow whole-genome sequencing (sWGS) using next-generation sequencing (NGS) for alternative POC cytogenomic analysis. Since female euploidy samples can include 69,XXX triploidy, additional QF-PCR was performed in these cases. Results: We here analyzed POC samples from miscarriages in 300 assisted reproductive technology (ART) pregnancies and detected chromosomal abnormalities in 201 instances (67.0%). Autosomal aneuploidy (151 cases, 50.3%) was the most frequent abnormality, consistent with prior conventional karyotyping data. Mosaic aneuploidy was detected in seven cases (2.0%). Notably, the frequency of triploidy was 2.3%, 10-fold lower than the reported frequency in non-ART pregnancies. Structural rearrangements were identified in nine samples (3%), but there was no case of segmental mosaicism. Conclusions: These data suggest that NGS-based sWGS, with the aid of QF-PCR, is a viable alternative karyotyping procedure that does not require cell culture. This method could also assist with genetic counseling for couples who undergoes embryo selection based on PGT-A data.

Effects of time-restricted feeding and type of food on fertility competence in female mice.

We assessed the effects of feeding regimen (ad libitum vs. time-restricted food access) and type of food (normal chow (NC: 12% fat) vs. moderately high calorie diet (mHCD: 31% fat)) on fertility competence of female mice. Mice fed mHCD had higher number of oocytes than mice fed NC. On the other hand, when mice were fed NC under time-restricted access to food (NT), the developmental rate to the blastocyst per number of normally fertilized ova was significantly decreased compared to others. The reactive oxygen species (ROS) level in oocytes increased in time-restricted food access and NC group. Transcriptome analysis of whole ovarian tissues from these mice showed a change in the cholesterol metabolism among the four groups. Time-restricted food access decreased serum LDL cholesterol level in both NC and mHCD groups. Moreover, the number of atretic follicles increased in NT mice compared to ad libitum food access mice. The present study shows that mHCD feeding increases the number of ovulated oocytes and that time-restricted feeding of NC impairs the developmental competence of oocytes after fertilization, probably due to the changes in serum cholesterol levels and an increase in the ROS content in oocytes.

MinION, a portable long-read sequencer, enables rapid vaginal microbiota analysis in a clinical setting.

BACKGROUND: It has been suggested that the local microbiota in the reproductive organs is relevant to women's health and may also affect pregnancy outcomes. Analysis of partial 16S ribosomal RNA (rRNA) gene sequences generated by short-read sequencers has been used to identify vaginal and endometrial microbiota, but it requires a long time to obtain the results, making it unsuitable for rapid bacterial identification from a small specimen amount in a clinical context. METHODS: We developed a simple workflow using the nanopore sequencer MinION that allows high-resolution and rapid differentiation of vaginal microbiota. Vaginal samples collected from 18 participants were subjected to DNA extraction and full-length 16S rRNA gene sequencing with MinION. RESULTS: The principal coordinate analysis showed no differences in the bacterial compositions regardless of the sample collection method. The analysis of vaginal microbiota could be completed with a total analysis time of approximately four hours, allowing same-day results. Taxonomic profiling by MinION sequencing revealed relatively low diversity of the vaginal bacterial community, identifying the prevailing Lactobacillus species and several causative agents of bacterial vaginosis. CONCLUSIONS: Full-length 16S rRNA gene sequencing analysis with MinION provides a rapid means for identifying vaginal bacteria with higher resolution. Species-level profiling of human vaginal microbiota by MinION sequencing can allow the analysis of associations with conditions such as genital infections, endometritis, and threatened miscarriage.

Effect of X-ray exposure during hysterosalpingography on capabilities of female germ cells.

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 < 16.23, mH-RAK: RAK ≥ 16.23). The number of oocytes retrieved, oocyte maturation, fertilization, and embryo development was compared among 3 groups. Further, multivariate analyses were performed to investigate the effect of X-ray exposure on laboratory outcomes in IVF. RESULTS: There was a statistically significant difference in the fertilization rate among 3 groups (Nil: 71.6%, L-RAK: 80.5%, mH-RAK: 78.3%). The good-quality blastocyst rate in mH-RAK (46.2%) was significantly higher than L-RAK (35.3%) and Nil (32.4%). Multivariate analyses revealed that X-ray exposure was associated with higher fertilization, higher blastocyst development, and higher good-quality blastocyst development rates with adjustment for patient age, BMI, ovarian stimulation types, and fertilization methods. Association between X-ray exposure and the number of oocytes retrieved, and oocyte maturation was not confirmed. CONCLUSIONS: The present study suggests that X-ray exposure of the female reproductive organs during HSG could enhance the potential of oocytes rather than adversely.

Mural granulosa cells support to maintain the viability of growing porcine oocytes and its developmental competence after insemination.

PURPOSE: To enhance the in vitro growth of porcine oocytes, we studied the effect of mural granulosa cells (MGCs) on the viability of oocytes attached to granulosa cells (oocyte-granulosa cell complexes, OGCs) that were obtained from early antral follicles. METHODS AND RESULTS: When OGCs were cultured with MGCs for 12 days, there were significant improvement (P < 0.05) in the robustness of gap junctional communication between the oocyte and the granulosa cells (82% vs. 59%), the survival rate of oocytes (57% vs. 39%), and the diameter of survived oocytes (118 µm vs. 112 µm). The rate of oocyte release of OGCs cultured with MGCs on the 12th day (1.9%) was significantly lower than that of OGCs cultured without MGCs (26%). Complete meiotic arrest was maintained in the group with MGCs (100%), while partial resumption of spontaneous meiosis was noticed in the absence of MGCs (10-19%). Furthermore, the presence of MGCs increased the oocyte maturation rate after maturation culture in both 12- and 14-day culture groups (P < 0.05, 85-88%) compared to OGCs cultured without MGCs (48-60%). MGCs also significantly improved the blastocyst formation rate (day 7) after ICSI (P < 0.05). CONCLUSIONS: The data of this study thus shows that the presence of MGCs during in vitro oocyte growth plays a crucial role in supporting the developmental competence of growing porcine oocytes attached to the granulosa cells via enhancement of their viability.