Hiroyuki Yazawa

Intracytoplasmic sperm injection using immobilized or motile human spermatozoon

OBJECTIVES To investigate the efficacy of the treatments for oocyte activation on the results of intracytoplasmic sperm injection using immobilized or motile human spermatozoa. DESIGN The protocol of intracytoplasmic sperm injection was divided into four groups according to the states of sperm used for microinjection and the treatment for oocyte activation. In group A, immobilized sperm is used. The oocyte is activated merely by aspiration of the cytoplasm into the pipette. In group B, immobilized sperm is used. Microinjected oocyte is treated with A23187. In group C, immobilized sperm is used. Electroporation is performed on the microinjected oocyte. In group D, motile sperm is used. The oocyte is activated merely by aspiration of the cytoplasm into the pipette. SETTING The Obstetrics and Gynecology Hospital, Fukushima Medical College. PATIENTS The subjects are the cases that had failed fertilization in standard IVF, cases of severe oligozoospermia, and cases of severe asthenozoospermia. RESULTS No difference was found between the groups as to the survival rate and fertilizing rates of oocytes after intracytoplasmic sperm injection. The cleavage rate of oocytes was high in order of group D, C, B, A. The cleavage rate for groups D, C, and B was significantly higher than group A. Cases of pregnancy were found in groups D and B. CONCLUSION Using motile sperm rather than immobilized sperm can be expected to produce better results in human ICSI. Activating oocytes positively is needed when immobilized sperm is used.

Pronuclear formation and cleavage of mammalian eggs after microsurgical injection of freeze-dried sperm nuclei

A study was conducted to evaluate the ability of mammalian oocytes to develop to the pronuclear stage and beyond if injected with freeze-dried sperm nuclei. The rate of development of hamster eggs to the pronuclear stage after microinjection of freeze-dried hamster sperm nuclei was 90%. The pronuclear formation rate of hamster eggs injected with freeze-dried human sperm nuclei was 85%. On the other hand, the rates for eggs injected with untreated sperm nuclei were 84% and 89% respectively. The survival rate of rabbit eggs microinjected with freeze-dried rabbit sperm nuclei was 64%, the fertilisation rate 56% and the cleavage rate 38%. The survival, fertilisation and cleavage rates of eggs injected with non-freeze-dried sperm nuclei were 78%, 45% and 34%, respectively. There was no difference between eggs injected with freeze-dried sperm nuclei and those injected with sperm nuclei that had not been freeze-dried. Of the viable rabbit eggs injected with freeze-dried rabbit sperm nuclei, 6% developed to the 6- to 8-cell stage 62 h after injection.

Spontaneous uterine rupture in the 33rd week of IVF pregnancy after laparoscopically assisted enucleation of uterine adenomatoid tumor

Although a uterine leiomyomectomy or adenomyomectomy is an accepted procedure to treat symptoms such as dysmenorrhea or hypermenorrhea to enhance fertility, the risk of future uterine rupture is a major concern for patients who become pregnant following these surgery. Although uterine rupture very rarely occurs, this is the most feared complication in pregnancy and is associated with a high rate of maternal and fetal morbidity and mortality.

Human round spermatids from azoospermic men exhibit oocyte-activation and Ca2+ oscillation-inducing activities.

During mammalian fertilization, intracellular Ca2+ oscillations are important for both oocyte activation and embryonic development. As the ability of round spermatids (ROS) to induce Ca2+ oscillations and oocyte activation is different between species, we examined Ca2+ oscillation- and oocyte activation-inducing abilities of human ROS originating from patients with non-obstructive azoospermia. Human ROS from 11 non-obstructive azoospermic patients were collected during their TESE-ICSI cycles. Following injection into mature unfertilized mouse oocytes, we examined the oocyte-activating and Ca2+ oscillation-inducing activities of ROS by using Ca2+ imaging and confocal laser scanning microscopy (mouse test). In these 11 cases, clinical TESE-ICSI using mature testicular spermatozoa was successful, with the exception of one case in which only one sperm-injected oocyte was not fertilized. The mean fertilization rate was 70.1% (40-100%); the mean cleavage rate was 97.9% (46/47). Two pregnancies were established from 10 transfer cycles (PR; 20%). When the ROS from these patients were injected into mouse oocytes, the ROS from all patients induced at least some intracellular Ca2+ oscillations (25-100%). In all patients, 40 out of 82 oocytes injected with ROS exhibited normal oscillation patterns of [Ca2+]i. Human spermatogenetic cells acquired oocyte-activating and Ca2+ oscillation-inducing abilities at the round spermatid stage, an earlier stage than found for rodent cells. These data indicate that human ROS might be useful for clinical treatments of non-obstructive azoospermic patients exhibiting mature spermatozoa in biopsied specimens.

Microinsemination of Rabbit Oocytes with Heat-Treated Sperm: Embryonic Development

A study was conducted to evaluate the ability of rabbit oocyte to develop to the pronuclear stage and subsequent development if injected with heat-treated sperm. The rate of fertilization of eggs after microinjection of cauda epididymal spermatozoa heated at 60 degrees C for 30 min was 36%; the rate of cleavage was 24%. The rate of fertilization after microinjection of heat-treated sperm nuclei was 43%; the rate of cleavage was 26%. On the other hand, the rate of fertilization of eggs injected with sperm without heat treatment was 43%, the rate of cleavage was 29%, and there was no difference between eggs microinjected with heat-treated spermatozoa or sperm nuclei and those not heat treated. Of the viable eggs injected with heat-treated sperm, 16% developed to the 6- to 8-cell stage 72 h after injection.

The oocyte activation and Ca2+ oscillation-inducing abilities of mouse and human dead (sonicated) spermatozoa.

In ICSI procedures, it is well known that the selection of viable (live) spermatozoa and certain types of immobilization prior to injection is very important for obtaining successful results, but unfortunately there are rare situations when only immotile spermatozoa are available (such as in severe asthenozoospermia or necrozoospermia). In such cases, failure of oocyte activation after ICSI often occurs and may be due to the lack of SOAF (sperm-borne oocyte activating factor) activity. In order to investigate the SOAF activities of dead spermatozoa, mouse and human spermatozoa were immobilized (killed by sonication), maintained in THF medium for varying time intervals (up to 72 h) and then injected into mature unfertilized mouse oocytes. Injected mouse oocytes were examined for their activation, development into blastocysts and Ca2+ responses by imaging and confocal laser scanning microscope. The rates of oocyte activation, blastocyst development and normal patterns of Ca2+ oscillation from the killed-sperm-injected oocytes decreased gradually in accordance with the maintenance interval between sonication and injection. For injection with mouse sonicated spermatozoa, the rate of normal Ca2+ oscillations declined first (after a 3 h maintenance interval) and then blastocyst development was gradually obstructed (after approx. 10 h). The oocyte activation-inducing ability of dead spermatozoa was maintained for a relatively long period, but began to decline after 20 h. The activation rates and Ca2+ response of the oocytes that were injected with human sonicated spermatozoa decreased earlier than those injected with mouse spermatozoa. Although the oocyte activation-inducing ability was maintained for a relatively long time after the death of the spermatozoa, embryo development into blastocysts and the rate of normal Ca2+ oscillations declined after a short maintenance interval between sonication and injection. The Ca2+ response seemed to be the most sensitive indicator for the evaluating the SOAF activity of dead (killed) spermatozoa.

Surgical outcomes of total laparoscopic hysterectomy with 2-dimensional versus 3-dimensional laparoscopic surgical systems

Three-dimensional (3D) laparoscopic surgical systems have been developed to account for the lack of depth perception, a known disadvantage of conventional 2-dimensional (2D) laparoscopy. In this study, we retrospectively compared the outcomes of total laparoscopic hysterectomy (TLH) with 3D versus conventional 2D laparoscopy. From November 2014, when we began using a 3D laparoscopic system at our hospital, to December 2015, 47 TLH procedures were performed using a 3D laparoscopic system (3D-TLH). The outcomes of 3D-TLH were compared with the outcomes of TLH using the conventional 2D laparoscopic system (2D-TLH) performed just before the introduction of the 3D system. The 3D-TLH group had a statistically significantly shorter mean operative time than the 2D-TLH group (119±20 vs. 137±20 min), whereas the mean weight of the resected uterus and mean intraoperative blood loss were not statistically different. When we compared the outcomes for 20 cases in each group, using the same energy sealing device in a short period of time, only mean operative time was statistically different between the 3D-TLH and 2D-TLH groups (113±19 vs. 133±21 min). During the observation period, there was one occurrence of postoperative peritonitis in the 2D-TLH group and one occurrence of vaginal cuff dehiscence in each group, which was not statistically different. The surgeon and assistant surgeons did not report any symptoms attributable to the 3D imaging system such as dizziness, eyestrain, nausea, and headache. Therefore, we conclude that the 3D laparoscopic system could be used safely and efficiently for TLH.

Long-term recurrence-free survival of a patient with advanced pure primary ovarian squamous cell carcinoma treated with dose-dense paclitaxel combined with carboplatin

We describe an extremely rare case of advanced pure primary ovarian squamous cell carcinoma (SCC), treated by adjuvant chemotherapy with dose-dense paclitaxel combined with carboplatin (dd-TC) plus the combination chemotherapy with irinotecan and cisplatin (CPT-P), with long-term recurrence-free survival. A 71-year-old woman complaining of lower abdominal pain was referred to our hospital and a 7-cm-diameter solid tumor was identified. She was diagnosed with a left ovarian tumor that was highly suspicious for malignancy based on ultrasonography, magnetic resonance imaging, and contrast-enhanced computed tomography. Bilateral salpingo-oophorectomy, low-anterior colon resection, and colostomy were performed. Intra- and post-operative histopathological diagnosis revealed International Federation of Gynecology and Obstetrics stage IIIc well-differentiated pure ovarian SCC. As adjuvant chemotherapy, 2 courses of dd-TC were administered, followed by 3 courses of CPT-P; the patient then underwent 4 additional courses of dd-TC. Both regimens were effective and there has been no recurrence or metastasis thus far in the 5 years since the operation.

A case of signet ring cell carcinoma of the ovary diagnosed after laparoscopic surgery

Signet ring cell carcinomas (SRCCs) of the ovary are rare. In general, primary SRCCs of the ovary are extremely rare; most are metastatic neoplasms. The gastrointestinal tract is the most likely primary site. We describe a rare case of an ovarian tumor removed with laparoscopic surgery in which the postoperative pathological diagnosis was SRCC of the ovary. The patient was a 39-year-old woman with a history of two vaginal deliveries. Her past medical and surgical histories were unremarkable. She complained of right lower abdominal pain. Transabdominal ultrasonography revealed a solid tumor of 9 cm in diameter adjacent to the right side of the uterus (Fig. 1A). A solid adnexal tumor was suspected. She was referred to our hospital for further evaluation and treatment. When uterine cervical cancer screening was performed 9 months previously, no abnormal cytology or tumors were detected. Magnetic resonance imaging (MRI) was also performed. T2-weighted imaging showed a solid tumor with uneven signal intensity adjacent to the body of the uterus (Fig. 1B). No ascites was detected by imaging. Consequently, a solid tumor arising from the right ovary such as a fibroma or thecoma was suspected. Tumor markers for epithelial ovarian cancer such as CA125, CA19-9, and CEA were all within normal range. Primary laparoscopic surgery was performed using three port insertions. There were no severe peritoneal adhesions. A small volume of yellow serous ascites was pooled in the pouch of Douglas, which was collected and examined intraoperatively. Cytology of the ascites fluid was class IIIa with atypical cells. The tumor was solid and 10 cm in diameter. It originated from the right ovary with good movability (Fig. 1C). The uterus and left ovary were morphologically normal in size and shape. We performed right salpingo-oophorectomy using the LigaSure VTM energy-sealing device (COVIDIEN, Japan). To avoid direct contact between the tumor and the port site, the resected ovarian tumor was packed into a specimen bag in the peritoneal cavity and removed through the 12-mm port site after it was cut into small pieces inside the bag (Fig. 1D and E). Although inflammation-like changes were observed on the pelvic peritoneum, adhesiolysis and peritoneal biopsy were not performed because there were no obvious findings suggestive of tumor dissemination and we did not suspect malignancy at that time (Fig. 1F).