Tomohisa Ugajin

Successful pregnancy after oocyte activation by a calcium ionophore for a patient with recurrent intracytoplasmic sperm injection failure, with an assessment of oocyte activation and sperm centrosomal function using bovine eggs

OBJECTIVE To report a successful pregnancy after intracytoplasmic sperm injection (ICSI) with artificial oocyte activation (AOA) on a patient whose fertilization rate after ICSI was extremely low; and to report on cytologic analyses of the fertilization failure (FF) eggs after ICSI and a biologic assessment of the sperm of this patient. DESIGN Case report with an assessment of gamete function. SETTING University hospital and an experimental laboratory. PATIENT(S) A couple with severe oligoasthenozoospermia, whose seventh attempt at ICSI ended in the failure. INTERVENTION(S) Cytologic analyses of FF eggs after ICSI, AOA after ICSI, and analyses of human sperm oocyte activation ability and centrosomal function. RESULT(S) Fertilization arrest after ICSI was observed in FF eggs at various stages of fertilization. After artificial oocyte activation by exposure to ionomycin, clinical pregnancy was confirmed, and a healthy baby was born. As assessed by heterologous ICSI of human sperm into bovine oocytes, there was no significant difference in the oocyte activation rates between the patients and control sperm, but the sperm centrosomal function was low in the patients sperm (48.5% vs. 69.6%). CONCLUSION(S) We report a successful pregnancy after ICSI with AOA using a calcium ionophore, after critical cytologic analyses of the FF eggs. Furthermore, sperm centrosomal function was low, indicating that sperms ability to process the events of fertilization after the oocyte activation was poor in this patient.

Aberrant behavior of mouse embryo development after blastomere biopsy as observed through time-lapse cinematography

OBJECTIVE To analyze whether blastomere biopsy affects early embryonal growth as observed through time-lapse cinematography. DESIGN Comparative prospective study between embryos in which a blastomere was removed and embryos in which a blastomere was not removed. SETTING An experimental laboratory of the university. MAIN OUTCOME MEASURE(S) We calculated the time between blastocele formation and the end of hatching, the time between the start and end of hatching, the number of contractions and expansions between blastocyst formation and the end of hatching, and the maximum diameter of the expanded blastocyst. RESULT(S) In blastomere removal embryos, compaction began at the six-cell stage instead of at the eight-cell stage. We also found that hatching was delayed in these embryos as compared with matched controls. Moreover, the frequency of contraction and expansion movements after blastocyst formation was significantly higher in the blastomere removal group as compared with the control group. Finally, the maximum diameter of the expanded blastocyst just before hatching was not significantly different between both groups. CONCLUSION(S) These findings suggested that blastomere removal has an adverse effect on embryonic development around the time of hatching. Thus, future developments in preimplantation genetic diagnosis and screening should involve further consideration and caution in light of the influence of blastomere biopsy on embryonal growth.

Functional assessment of centrosomes of spermatozoa and spermatids microinjected into rabbit oocytes

Although intracytoplasmic sperm injection (ICSI) is a widely used assisted reproductive technique, the fertilization rates and pregnancy rates of immature spermatids especially in round spermatid injection (ROSI) remain very low. During mammalian fertilization, the sperm typically introduces its own centrosome which then acts as a microtubule organizing center (MTOC) and is essential for the male and female genome union. In order to evaluate the function of immature germ cell centrosomes, we used the rabbit gamete model because rabbit fertilization follows paternal pattern of centrosome inheritance. First, rabbit spermatids and spermatozoa were injected into oocytes using a piezo‐micromanipulator. Next, the centrosomal function to form a sperm aster was determined. Furthermore, two functional centrosome proteins (γ‐tubulin and centrin) of the rabbit spermatogenic cells were examined. Our results show that the oocyte activation rates by spermatozoa, elongated spermatids, and round spermatids were 86% (30/35), 30% (11/36), and 5% (1/22), respectively. Sperm aster formation rates after spermatozoa, elongated spermatids, and round spermatids injections were 47% (14/30), 27% (3/11), and 0% (0/1), respectively. The aster formation rate of the injected elongating/elongated spermatids was significantly lower than that of the mature spermatozoa (P = 0.0242). Moreover, sperm asters were not observed in round spermatid injection even after artificial activation. These data suggest that poor centrosomal function, as measured by diminished aster formation rates, is related to the poor fertilization rates when immature spermatogenic cells are injected. Mol. Reprod. Dev. 76: 270–277, 2009.

Risk factors for recurrence and re-recurrence of ovarian endometriomas after laparoscopic excision.

Aim:  Since ovarian endometrioma is frequently diagnosed in women of reproductive age, laparoscopic excision of the endometrioma is performed for most cases. However, endometriomas frequently recurs even after repeated surgical procedures. The aim of our study is to identify risk factors for recurrence and re‐recurrence of endometriomas after the first and second laparoscopic excision.

Different embryonic development after blastomere biopsy for preimplantation genetic diagnosis, observed by time-lapse imaging

Using time-lapse imaging, we found different behavior of mouse embryonal development after blastomere biopsy for preimplantation genetic diagnosis. Blastomere removal has effects on the developmental behavior of the mouse embryo, including speed of growth, contraction and expansion movements, and hatching.

Total Laparoscopic Conservative Surgery for an Intramural Ectopic Pregnancy

A 38-year-old woman, gravida 3, para 1 with a history of a left salpingectomy for an ectopic pregnancy was admitted for treatment of a presumed ectopic pregnancy. Transvaginal sonography revealed an ill-defined gestational sac and fetal heart beat within the fundal myometrium adjacent to the left cornua. Laparoscopy was performed for a suspected left cornual pregnancy or intramural pregnancy. A cystic mass 3 cm in diameter was visible within the fundal myometrium. Total laparoscopic removal of the gestational sac was performed, and the uterus was preserved. Pathologic evaluation of the excised mass demonstrated chorionic villi involving the myometrium. In the literature, only one other case describing the laparoscopic removal of an intramural pregnancy has been reported. However, in the prior report, the patient still required hysterectomy after conservative surgery. Therefore, this is the first report of the successful treatment of an intramural pregnancy exclusively with laparoscopy.

Microtubule organization during human parthenogenesis.

In human fertilization, the sperm centrosome plays a crucial role as a microtubule organizing center (MTOC). We studied microtubule organization during human parthenogenesis, which occurs when a human egg undergoes cleavage without a sperm centrosome. Multiple cytoplasmic asters were organized in the human oocyte after parthenogenetic activation, indicating that multiple MTOC are present in the human oocyte cytoplasm and function like a human sperm centrosome during parthenogenesis.

The shape of the sperm midpiece in intracytoplasmic morphologically selected sperm injection relates sperm centrosomal function

PurposeTo evaluate whether the morphology of the sperm midpiece observed by high magnification microscopy relates to sperm centrosomal function.MethodsSperm selected by conventional microscopy were defined as controls. By high magnification microscopy, sperm with straight midpieces were defined as Group 1, while those with tapering midpieces were defined as Group 2. Heterologous ICSI of human sperm into bovine oocytes was used to assess human sperm centrosomal function and analysis of sperm aster formation.ResultsThe total rate of sperm aster formation was 80.5% in Group 1, which was significantly higher (P < 0.05) than the rate of 33.3% seen for Group 2. Furthermore, sperm aster formation rates tended to be higher for Group 1 than for the controls.ConclusionsThis study demonstrates improvement of sperm aster formation rates by selecting sperm on the basis of midpiece morphology. The injection of selected sperm bearing morphologically straight midpieces may contribute to improved expression of sperm centrosomal function, providing a positive effect on fertilization after ICSI.

A novel culture system for mouse spermatid maturation which produces elongating spermatids capable of inducing calcium oscillation during fertilization and embryonic development.

PurposeTo establish an in vitro culture system for mouse round spermatids that models spermiogenesis and enables the assessment of oocyte activation ability.MethodsRound spermatids and Sertoli cells were isolated from testicular tissues of B6D2F1 male mice and co-cultured in the presence of testosterone and recombinant FSH. Cultured spermatids were examined for morphology and condensation of nuclei, fertilization and development rate, and Ca2+ oscillation pattern after ICSI.ResultsThe cultured spermatids elongated and resembled normal elongating spermatids in terms of both morphology and nuclear condensation. No significant differences in fertilization and development rates were observed between fresh and cultured elongating spermatids. Moreover, cultured spermatids showed similar Ca2+ oscillation patterns to fresh elongating spermatids during an initial stage in oocyte activation.ConclusionsThese data suggest that a co-culture system of spermatids and Sertoli cells, supplemented with testosterone and recombinant FSH, supports normal differentiation of round spermatids into elongating spermatids, as assessed by their morphology, nuclear condensation, and oocyte activation ability.

Development of human Graafian follicles following transplantation of human ovarian tissue into NOD/SCID/gammac null mice.

Problem  Transplantation of human ovarian cortex into host mice may permit various kinds of challenges in reproductive medicine. A novel immunodeficient mouse strain (NOD/SCID/γcnull: NOG) has been developed as a host of transplantation of human tissue.