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Purpose Following reports of an increase in implantation and pregnancy continuation rates by a higher percentage of Lactobacillus in the intrauterine microbiota, it has received attention in infertility treatment. This study aimed to examine Japanese women for intrauterine microbiota. Methods The clinical background factors in women that influence the abundance of Lactobacillus in the bacterial microbiota were examined. We included 147 patients (31 and 116 in the follicular and luteal phase, respectively), from June 2018 to June 2020, who underwent their first intrauterine microbiota test and had not used antibiotics for at least 4 weeks before the test. In the luteal phase, we compared the background factors of women in cases with 90% or more and less than 90% of Lactobacillus. Differences in the intrauterine microbiota were examined during the follicular and luteal phases. Results The proportion of Lactobacillus tended to be low among women aged 36 years and older with a history of childbirth (P = 0.0631). Some bacteria were only detected during the follicular and luteal phases, and the bacterial microbiota may change during the menstrual cycle. Conclusion Bacterial microbiota in the uterus may differ between the follicular and luteal phases. Furthermore, it was shown that the rate of Lactobacillus may be lower in women (older than 36 years) who had given birth, indicating that intrauterine microbiological testing may be considered for these women in clinical practice. Lay summary Good implantation and pregnancy continuation rates have been reported when the proportion of the bacteria Lactobacillus is high in the uterus (intrauterine) bacterial population (microbiota). In this study, we assessed whether the clinical background of Japanese women (age, history of pregnancy and childbirth, and presence of gynecological or hormonal disorders) affect the proportion of intrauterine microbiota. Intrauterine samples were collected and sequenced to evaluate the intrauterine microbiota and the composition ratio of each bacterium. Comparing the percentage of Lactobacillus in the latter phase of the menstrual cycle with the clinical background, it was found that the percentage tended to be lower in women with a history of childbirth. We compared the intrauterine microbiota between the first phase and latter phase of the menstrual cycle and revealed that it may differ between the two phases. Advances in the development of criteria for assessing intrauterine microbiota are expected. more...

In the field of in vitro fertilization (IVF), useful markers for the prediction of successful implantation for oocyte or embryo selection are essential. It has been reported that sHLA-G (sHLA-G1/HLA-G5) could be detected in the supernatant of the fertilized embryo and in follicular fluid samples (FFs), and that the presence of sHLA-G was related to successful implantation. If sHLA-G could be used as a marker of oocyte selection from multiple FFs, oocytes could be selected without physical contact, thus reducing the likelihood of damage. To investigate the potential for sHLA-G as a marker of oocyte selection from multiple FFs in one patient, protein levels of total protein, sHLA-G, and sHLA-I (sHLA-A, B, and C) were examined in FFs. The variation among multiple FFs in total protein level and sHLA-G level was not related to successful pregnancy. The average sHLA-I levels did not differ in the successful implantation and unsuccessful implantation groups, indicating that sHLA-I levels were not related to successful pregnancy. Furthermore, sHLA-G in FFs was not detected by western blotting, despite being detected by ELISA, while sHLA-I was detected by both ELISA and western blot. These data suggest that sHLA-G in FF might not be a useful marker for oocyte selection as measurements of sHLA-G were inconsistent and there was no association with successful pregnancy. Further, more rigorously tested ELISA systems for detecting sHLA-G in body fluids are necessary before the utility of sHLA-G for diagnosis can be established. more...

Tetraspanin CD9 is essential for sperm-egg fusion and also contributes to uterine repair through microexosome formation. Microexosomes share CD9 with exosomes and are released from eggs and uterine epithelial cells. However, the mechanism for the formation of microexosomes remains unknown. To address this issue, we examined membrane localization and extracellular release of CD9 proteins using uterine epithelial cells and secretions in mice and humans. In mice, CD9 localized predominantly on the basal region of the plasma membrane and relocated to the apical region upon embryo implantation. Furthermore, extracellular CD9 proteins were detected in uterine secretions of mice and women undergoing infertility treatment, but were below detectable levels in supernatants of pluripotent stem cells. Ultrastructural analysis demonstrated that membrane projections were shortened and the number of mitochondria was reduced in uterine epithelial cells lacking Cd9 genes. Our results suggest that CD9 repositioning and release affect both membrane structures and mitochondrial state in the uterus, and contribute to female fertility. more...

Spermatozoa used for intracytoplasmic sperm injection (ICSI) are selected based on their motility and morphology. To explore other methods for selecting better spermatozoa, we developed a spermatozoa-sorting method using a physiologically natural selection system involving penetration into cervical mucus (CM). In addition, we analyzed the spermatozoa that penetrated the CM (CM-penetrating spermatozoa). The results were as follows. The CM-penetrating spermatozoa traveled a longer distance with better linear motility than spermatozoa in semen. Also, in comparison with spermatozoa obtained by density gradient centrifugation (DGC) and the swim-up method, the proportion of spermatozoa with normal morphology was higher, although, the proportion of spermatozoa with the head vacuole did not change. No DNA fragmentation was detected in the CM-penetrating spermatozoa. This method has several advantages. The technical procedure is simple and easy. Physical damage to spermatozoa is reduced because it does n... more...

In cases of testicular sperm extraction sperm aspiration and severe oligozoospermia for which intracytoplasmic sperm injection (ICSI) is indicated, there are cases where motile sperm are difficult to obtain even from collected semen. In cases when little or no motile sperm is obtainable from prepared sperm suspension. We stimulated sperm motility using pentoxifylline and then selected live sperm. The clinical outcomes of recovery of live sperm were compared with cases in which motile sperm was obtainable. Fertilization and embryo recovery rates of the pentoxifylline group were significantly lower than those of the control group. However, the pregnancy, implantation, and abortion rates of the pentoxifylline group were not significantly different from those of the control group, suggesting a favorable outcome if a viable embryo is implanted. Moreover, no abnormal findings were found in babies subsequently born. Considering that the important point of ICSI is to inject live sperm, pentoxifylline tr... more...

In ICSI, sperm is selected based on its motility and shape under 200–400× magnification. However, sperm morphology, especially the existence of vacuoles, may relate to sperm nuclear DNA fragmentation. Bartoov et al. reported the application of sperm selection under high (1,000–6,000×) magnification, which they referred to as IMSI (intracytoplasmic morphologically-selected sperm injection). In our initial trials, the fertilization, blastocyst development, pregnancy and miscarriage rates of 115 single blastocyst transfer cycles with IMSI or ICSI were 84.0% vs. 75.5%, 47.5% vs. 43.7%, 50.0% vs. 47.4% and 10.0% vs. 11.0%, respectively. Further studies are required to establish the systems and clarify the indications for IMSI. more...

In recent years, the number of patients receiving in vitro fertilization (IVF) has been increasing, though the rate of successful implantations has remained at 10-20%. A major goal of this procedure is to afford the ability to select embryos with the most potential for implantation and development. Previous studies claimed to have detected soluble HLA-G (sHLA-G) protein in culture supernatant from 2 to 3-day embryos using ELISA methods, and concluded that sHLA-G protein levels were associated with successful implantation. This result, if substantiated could provide an important tool for IVF. In this study, we have re-examined these experiments by attempting to detect sHLA-G in the medium from 2 to 3-day embryos (84 samples) and 4 to 6-day embryos (25 samples) in which a part of blastocyst has started to differentiate into trophoblasts. Using a highly specific and sensitive ELISA, no sHLA-G protein was detectable in any sample, despite the fact that 27 of the 109 samples were from successfully implanted embryos. These results indicate that 2-6-day embryos do not secrete sHLA-G detectable by ELISA, and therefore that sHLA-G in culture medium is not a useful for successful implantation at this stage of development. more...

Background Recently, the concept of recurrent implantation failure (RIF) in assisted reproductive technology has been enlarged. Chronic uterine inflammation is a known cause of implantation failure and is associated with high matrix metalloproteinase (MMP) activity in uterine cavity flushing. MMP activity of women with RIF has been reported to be higher than that of fertile women. In the present retrospective study we evaluated the efficacy of treatment for high MMP activity in the uterine cavity of patients with RIF. Methods Of the 597 patients recruited to the study, 360 patients underwent MMP measurements and 237 patients did not (control group). All patients had failed to become pregnant, despite at least two transfers of good-quality embryos. Gelatinase MMP-2 and MMP-9 activity in uterine flushing fluid was detected by enzymology (MMP test). All samples were classified into two groups (positive or negative) based on the intensity of the bands on the enzyme zymogram, which represents the degree of MMP activity. Patients who tested positive on the initial test were treated for 2 weeks with a quinolone antibiotic and a corticosteroid, and subsequently underwent a second MMP test. Negative results on the second MMP tests after treatment and subsequent rates of pregnancy and miscarriage were used to evaluate the efficacy of treatment. Data were analyzed by the Mann–Whitney U-test and the chi-square test. Results Of the patients who underwent the MMP test, 15.6% had positive results (high MMP activity). After treatment, 89.3% of patients had negative results on the second MMP test. These patients had a significantly better pregnancy rate (42.0%) than the control group (26.6%), as well as a lower miscarriage rate (28.5% vs 36.5%, respectively). Conclusions A 2-week course of antibiotics and corticosteroids effectively improves the uterine environment underlying RIF by reducing MMP activity. more...

We investigated whether the aged oocytes maintain the ability to fuse with spermatozoa when the oocyte investment is removed. In the first study, 87 two-days-old oocytes provided from IVF-ET programme were treated with pronase to dissolve the zona pellucida. The oocytes were then inseminated with 100, 10 or 1 per microliters sperm and the incidence of monospermic and polyspermic fertilization was determined. In the second study 100 one-day-old unfertilized oocytes obtained from 18 IVF patients with "zero fertilization" at initial insemination were treated in the same way to either remove or thinning of the zona pelucida. They were then re-inseminated with 10 motile sperm per microliter of the husband. The incidence of monospermic and polyspermic fertilization were 0.0% (0/13) and 61.6% (8/14), respectively, with a sperm count of 100/microliters, 7.3% (3/41) and 31.6% (13/41) with 10/microliters, and 14.2% and 11.9% (5/33) with 1/microliter. The incidence of monospermic and polyspermic fertilization with reinsemination of one day old oocytes were 12.5% (3/24) and 25.0% (6/24) after zona removal and were 18.3% (9/49) and 10.2% (5/49) after zona thinning. The incidence of fertilization by zona removal was significantly higher than the results of conventional reinsemination (p < 0.001). It appeared that human aged oocytes maintain the ability to fuse spermatozoa after even two days. A higher rate of monospermic fertilization was obtained by carefully controlling the number of spermatozoa for insemination. We concluded that zona manipulation might improve fertilization in vitro in case of severe male factor infertility. more...

Micromanipulation was used for creating a small opening in the zona pellucida without damaging the ooplasm of oocytes. This was applied in mouse in vitro fertilization as a model for inseminating oocytes with low sperm concentrations. It was found that the opening in the zona did not influence the fertilization rate at 10(5) sperm/ml, but when the sperm concentration was decreased to 10(4)/ml, the oocytes containing the break had a significantly higher fertilization rate and blastocyst development, compared with their intact controls. The zona cracking procedure did not increase parthenogenetic activation nor polyspermic fertilization, although it did promote early hatching of some blastocysts. The birthrate of normal offspring was not affected by exposing oocytes to the zona opening process. more...