Yasuyuki Mio

Time-lapse cinematography of dynamic changes occurring during in vitro development of human embryos.

OBJECTIVE The purpose of this study was to clarify developmental changes of early human embryos by using time-lapse cinematography (TLC). STUDY DESIGN For human ova, fertilization and cleavage, development of the blastocyst, and hatching, as well as consequent changes were repeatedly photographed at intervals of 5-6 days by using an inverse microscope under stabilized temperature and pH. Photographs were taken at 30 frames per second and the movies were studied. RESULTS Cinematography has increased our understanding of the morphologic mechanisms of fertilization, development, and behavior of early human embryos, and has identified the increased risk of monozygotic twin pregnancy based on prolonged incubation in vitro to the blastocyst stage. CONCLUSION Using TLC, we observed the fertilization of an ovum by a single spermatozoon, followed by early cleavages, formation of the morula, blastocyst hatching, changes in the embryonic plates, and the development of monozygotic twins from the incubated blastocysts.

Subtle rise in serum progesterone during the follicular phase as a predictor of the outcome of in vitro fertilization

OBJECTIVE To investigate the effects of subtle rises in serum progesterone (P) during the follicular phase on the outcome of in vitro fertilization and embryo transfer (IVF-ET). DESIGN, PATIENTS One hundred one patients underwent IVF-ET for 170 cycles and were stimulated with a combination of clomiphene citrate and human menopausal gonadotropin. Based on their hormonal data, we divided the patients into two groups: those who had a cycle with an increase in serum P concentration (1.0 to 2.0 ng/mL) that was not associated with a pituitary LH release (subtle P rise) and those who had a cycle without any increase in serum P concentration (no P rise). MAIN OUTCOME MEASURES The daily serum estradiol (E2) concentration and the results of IVF-ET (number of developed and collected oocytes, rates of mature oocytes, fertilization, and pregnancy) were compared between the two groups. RESULTS Subtle P rises were observed in 31.7% (32/101) of the patients and 20.5% (36/170) of the cycles evaluated during the IVF-ET programs. A significantly higher serum E2 concentration (P less than 0.001) and a greater number of developed and collected oocytes (P less than 0.001 and P less than 0.05, respectively) also were observed in those cycles with a subtle P rise. The rates of mature oocyte formation and fertilization were significantly lower in cycles with a subtle P rise (P less than 0.001 and P less than 0.05, respectively). A lower pregnancy rate was observed in cycles with a subtle P rise, and all 12 ongoing pregnancies occurred only in cycles with a no P rise. CONCLUSION These results suggest that the development of an increased number of follicles may not necessarily improve the outcome of IVF-ET and that the measurement of serum P may be a better predictor for successful pregnancy.

Luteinized unruptured follicle in the early stages of endometriosis as a cause of unexplained infertility.

OBJECTIVE We attempted to clarify the relationship between luteinized unruptured follicle, which occurs in the early stages of endometriosis, and unexplained infertility. STUDY DESIGN Seventy patients who had unexplained infertility were reviewed. RESULTS Laparoscopic examination showed that 47 patients (67%) had endometriosis; of these, 40 (85%) had minimal or mild disease. The incidence of luteinized unruptured follicle was higher (p < 0.05) in patients who had endometriosis (35%/patient and 25%/cycle) compared with patients who did not have endometriosis (11%/patient and 7%/cycle). Degenerated oocyte cumuli were collected in 6 (43%) of 14 luteinized unruptured follicles diagnosed by transvaginal ultrasound. CONCLUSIONS These results show that luteinized unruptured follicle is common in patients who have mild or minimal endometriosis and that it may be one of the causes of endometriosis-associated infertility. Transvaginal ultrasound-guided follicular puncture of luteinized unruptured follicle during the mid luteal phase may be useful in establishing a definitive diagnosis of luteinized unruptured follicle.

Transvaginal ultrasound-guided follicular aspiration in the management of anovulatory infertility associated with polycystic ovaries *

STUDY OBJECTIVE To investigate whether or not transvaginal ultrasound (US)-guided follicular aspiration can effectively induce ovulation and facilitate pregnancy in anovulatory patients with polycystic ovaries (PCO). DESIGN Eight patients with polycystic ovarian disease (PCOD) and 10 patients with PCO were participants who failed to ovulate by the medical therapies. Most of persistent follicles were punctured, and their contents were thoroughly aspirated during the midluteal phase. The same ovarian stimulation regimen as used in the previous cycles were administered in the cycles after the aspiration. MAIN OUTCOME MEASURES Evidence of ovulation and a subsequent pregnancy was ultrasonically monitored after the aspiration, and the responsiveness of pituitary gonadotropins to gonadotropin-releasing hormone was tested in these patients. RESULTS The ovulation rates were 87.5% per patient, 52.6% per cycle monitored in PCOD patients and 100% per patient, 63.3% per cycle monitored in PCO patients, respectively. Half of the patients both with PCOD and PCO achieved pregnancy after the aspiration. A significant decrease (P less than 0.05) of the basal and peak levels of serum luteinizing hormone was observed after the aspiration. CONCLUSIONS The US-guided follicular aspiration seems to be a new surgical method for treating anovulatory patients with PCO.

Influence of sexual stimulation on sperm parameters in semen samples collected via masturbation from normozoospermic men or cryptozoospermic men participating in an assisted reproduction programme.

To evaluate the influence of sexual stimulation via sexually stimulating videotaped visual images (VIM) on sperm function, two semen samples were collected from each of 19 normozoospermic men via masturbation with VIM. Two additional samples were collected from each man via masturbation without VIM. The volume of seminal plasma, total sperm count, sperm motility, percentage of morphologically normal spermatozoa, outcome of hypo‐osmotic swelling test and zona‐free hamster oocyte sperm penetration assay, and markers of the secretory function of prostate were significantly larger in semen samples collected via masturbation with VIM than masturbation without VIM. The improved sperm parameters in the samples collected via masturbation with VIM may reflect an enhanced prostatic secretory function and increased loading of the vas deferens at that time. In a similar protocol, two semen samples were collected via masturbation with VIM from each of 22 non‐obstructed azoospermic men. Semen samples from these men had been occasionally positive in the past for a very small number of spermatozoa (cryptozoospermic men). Two additional samples were collected from each cryptozoospermic man via masturbation without VIM. The volume of seminal plasma, total sperm count, sperm motility, and a marker of the secretory function of prostate were significantly larger in semen samples collected via masturbation with VIM. Fourteen out of the 22 men were negative for spermatozoa in both samples collected via masturbation without VIM. These men demonstrated spermatozoa in both samples collected via masturbation with VIM. Six men with immotile spermatozoa in both samples collected via masturbation without VIM exposed motile spermatozoa in both samples collected via masturbation with VIM. High sexual stimulation during masturbation with VIM results in recovery of spermatozoa of greater fertilizing potential both in normozoospermic and cryptozoospermic men. The appearance of spermatozoa after masturbation with VIM in the vast majority of cryptozoospermic men is of clinical significance in programmes applying intracytoplasmic sperm injections for the management of severe male infertility and obviates the need for testicular biopsy.

Use of a highly sensitive quantitative telomerase assay in intracytoplasmic sperm injection programmes for the treatment of 47,XXY non-mosaic Klinefelter men

Summary.  We evaluated the role of the sensitive quantitative telomerase assay (SQTA) in the management of men with non‐mosaic Klinefelters syndrome (KS). Diagnostic testicular biopsy (DTB) was performed in 24 men with KS. A part of the DTB was stained and the remaining fragment was processed for the SQTA. After 3–18 months, a therapeutic testicular biopsy (TTB) was performed in the same testicle and the recovered specimens were processed to identify spermatozoa. Men with a SQTA outcome equal to 0.00 Units μg−1 protein (n=7) demonstrated therapeutic testicular biopsy material that was negative for spermatogenic cells. In five men with a SQTA outcome of 8.11–38.03 Units μg−1, the most advanced germ cell was the spermatogonium/primary spermatocyte. In the remaining 12 men, the most advanced spermatogenic cell in the TTB was the spermatozoon. In these men, the SQTA outcome was equal to 25.76–92.68 Units μg−1 protein. Using 39.00 Units  μg−1 protein as a cut‐off value, the accuracy of the SQTA in identifying men positive for spermatozoa was 91.6%. It appears that the SQTA has a role for identifying non‐mosaic KS men who have testicular spermatozoa.

The Importance of the Hypoosmotic Swelling Test and Acrosin Activity Assay for Identifying Subpopulations of Idiopathic Infertile Men

Semen samples collected from fertile donors (n = 13) and pooled samples from idiopathic infertile men (n = 19) were used in this study. Measurements of the total sperm acrosin activity and the hypoosmotic swelling test (HOST) were performed in all the samples. The percentage of swollen spermatozoa and acrosin profiles were significantly lower in the infertile men than in the fertile donors. Considering the lowest values of the outcome of the HOST and the acrosin activity assay in the group of fertile men as the lowest normal values, it was proven that HOST and acrosin activity assay could identify subpopulations of infertile men of 37 and 26%, respectively. The results tend to support the employment of the HOST and the acrosin activity assay in the evaluation of idiopathic infertile men.

Measurement of uptake and incorporation of nucleic acid precursors by preimplantation mouse embryos after development in vivo and in vitro

PurposeTo assay DNA and RNA synthesis by developing mouse embryos in vitro and in vivo, we measured the uptake and incorporation of3 H-thymidine and3 H-uridine by morulae and blastocysts. We also evaluated the effect of adding EDTA to the culture medium on the uptake and incorporation of nucleic acid precursors by blastocysts.ResultsThymidine and uridine incorporation increased after morulae developed into early blastocysts both in vitro and in vivo. However, the rates of uptake and incorporation were significantly lower by embryos grown in vitro than by those grown in vivo. The ratios of incorporation to total uptake were similar in embryos grown in vitro and in vivo. EDTA (100 µM) added to the culture medium significantly increased the incorporation of uridine into RNA by blastocysts grown in vitro (P <0.01) but did not increase the total uptake of uridine.ConclusionThese observations showed that both DNA and RNA synthesis increased during the early development of preimplantation embryos and that those activities were reduced in embryos undergoing development in vitro. The results also suggested that through the mechanism of EDTA effect in embryo culture remains unknown, it appeared to reduce the retardation of RNA synthesis by embryos cultured in vitro through a selective stimulation of uridine incorporation.

Timing of progesterone rise around a surge in endogenous luteinizing hormone or the injection of human chorionic gonadotropin in controlled ovarian stimulation for in vitro fertilization

SummaryOf 70 treatment cycles in 59 patients, the endogenous LH surge occurred in 19 cycles (27%) before fulfilling our criteria for hCG administration. Timed injections of hCG were administered in 31 cycles (44%). Twenty cycles were canceled. The elevation of P (>1.0 ng/ml) did not always occur before or during the onset of LH surge. Pregnancy occurred only during those cycles in which an elevated P level was observed during or 12 hr after the LH surge or of hCG administration. It is concluded that the timing of the P elevation as well as the P level itself has major physiological importance in controlled ovarian stimulation for IVF.

Observation of human embryonic behavior in vitro by high-resolution time-lapse cinematography

Assisted reproductive technology (ART) has yielded vast amounts of information and knowledge on human embryonic development in vitro; however, still images provide limited data on dynamic changes in the developing embryos. Using our high-resolution time-lapse cinematography (hR-TLC) system, we were able to describe normal human embryonic development continuously from the fertilization process to the hatched blastocyst stage in detail. Our hR-TLC observation also showed the embryonic abnormality of a third polar body (PB)-like substance likely containing a small pronucleus being extruded and resulting in single-pronucleus (1PN) formation, while our molecular biological investigations suggested the possibility that some 1PN embryos could be diploid, carrying both maternal and paternal genomes. Furthermore, in some embryos the extruded third PB-like substance was eventually re-absorbed into the ooplasm resulting in the formation of an uneven-sized, two-PN zygote. In addition, other hR-TLC observations showed that cytokinetic failure was correlated with equal-sized, multi-nucleated blastomeres that were also observed in the embryo showing early initiation of compaction. Assessment combining our hR-TLC with molecular biological techniques enables a better understanding of embryonic development and potential improvements in ART outcomes.