Koichi Kyono

Potential indications for ovarian autotransplantation based on the analysis of 5,571 autopsy findings of females under the age of 40 in Japan.

Ovarian cryopreservation and autotransplantation could be of potential value for preservation of fertility in the patients with various malignancies. Ovarian tissue should be cryopreserved actively for fertility preservation, but stored tissue should be autotransplanted with much caution until reliable methods are established to detect minimal residual disease in grafts in precise and reproducible manners.

Freezing of Human Immature Oocytes Using Cryoloops with Taxol in the Vitrification Solution

AbstractPurpose: In human frozen immature oocytes, there has been little successful delivery. We examined the feasibility of vitrification solution including Taxol, cytoskeltal stabilizer. Methods: We set four experimental groups that immature oocytes has cumulus cells or not, or including Taxol or not in the vitrification solution. Frozen–thawed oocytes have been performed IVM, ICSI, and IVC. Results: There were no significant differences in survival, maturation, and fertilization rate, respectively. However, in the group enveloped by cumulus cells and including Taxol in the vitrification solution, one embryo was developed to blastocyst. Conclusions: Our results showed that using vitrification solution with Taxol proved so effective.

Seven pregnancies and deliveries from non-mosaic Klinefelter syndrome patients using fresh and frozen testicular sperm.

AbstractPurpose: The aim of this study was to investigate the feasibility of using frozen-thawed testicular sperm as well as the timing of testicular sperm extraction (TESE) in patients with non-mosaic Klinefelter syndrome. Methods: Intracytoplasmic sperm injection (ICSI) was performed in six of 17 (35%) patients whose sperm was recovered by TESE. Multiple biopsies of both testes were performed on the day of oocyte retrieval in all but one of the six patients. Results: Seven pregnancies and deliveries were achieved in five couples, and one couple was unsuccessful. Five pregnancies were achieved using fresh motile sperm, and two were achieved using frozen-thawed sperm. Sperm cryopreservation was not possible in one of the five couples because of the small number of recovered sperm, and possible in four other couples for subsequent ICSI. One woman whose husband had TESE performed prior to ovarian stimulation did not become pregnant. This may be due to the attainment of only a few immotile sperm following the frozen-thawed procedure. Conclusion: The outcome of ICSI using fresh or frozen-thawed testicular sperm in patients with non-mosaic Klinefelter syndrome was identical; however, TESE should be performed on the day of oocyte retrieval until such time as a procedure with a higher sperm yield from TESE is available. Moreover, an improved recovery procedure after cryopreservation-thawing of a single spermatozoon must be developed.

Fresh motile testicular sperm retrieved from nonobstructive azoospermic patients has the same potential to achieve fertilization and pregnancy via ICSI as sperm retrieved from obstructive azoospermic patients

OBJECTIVE To compare the fertilization and pregnancy rates using fresh testicular sperm between nonobstructive azoospermic (NOA) patients and obstructive azoospermic (OA) patients. DESIGN We evaluated sperm quality of testicular sperm retrieved by microdissection testicular sperm extraction (MD-TESE) in NOA patients and compared the fertilization rate and pregnancy rate via intracytoplasmic sperm injection (ICSI) between NOA and OA patients. SETTING Private hospital-based infertility research laboratory. PATIENT(S) A total of 58 couples in which the husband was diagnosed with azoospermia. INTERVENTION(S) Analytic examination of the outcomes and description of the NOA cases achieving pregnancies; oocyte retrieval and testicular sperm retrieval were performed simultaneously. MAIN OUTCOME MEASURE(S) Comparison of fertilization rate and pregnancy rate at the first ICSI attempt. RESULT(S) Testicular sperm were retrieved from 17 of 40 NOA patients and 18 of 18 OA patients. Motile sperm were successfully retrieved from 16 of the 17 NOA patients. There was no significant difference in fertilization rate and pregnancy rate between OA and NOA cases. Of the 17 NOA patients, nine pregnancies were achieved using fresh motile testicular sperm. CONCLUSION(S) Fresh motile testicular sperm retrieved from NOA patients may have the same potential to achieve fertilization and pregnancy as sperm retrieved from OA patients. The MD-TESE technique might contribute to the high retrieval rate of fresh motile testicular sperm even in NOA patients.

Features of constitutive gr/gr deletion in a Japanese population

BACKGROUND The relationship between male infertility and gr/gr deletions that remove multiple genes of the Y chromosome varies among countries and populations. The aim of this study was to investigate the association between gr/gr deletions and spermatogenic phenotype in fertile and infertile Japanese men. METHODS The subjects were screened by sequence-tagged site (STS) analysis to detect gr/gr deletions, and haplogroups were assigned using eight highly informative markers. In total, 395 infertile men and 377 fertile men (controls) participated in our study. Of the 772 subjects, 260 individuals carried confirmed gr/gr deletions and were used in further analysis of deletion subtype and gene copy number, specifically loss and gain of CDY1 and DAZ copies. These 260 subjects were divided into a control group (n = 131) all with normozoospermia, and an infertile group (n = 129) with 89 infertile subjects exhibiting azoospermia (absence of sperm) and 40 exhibiting oligozoospermia (reduced sperm concentration). RESULTS There were gr/gr deletions in 33.7% (260/772) of all subjects and the deletions were widespread in haplogroup D (86.2%). There were no significant differences in the frequency of gr/gr deletions between the infertile and control groups. The gr/gr deletion subtypes were not distributed randomly among haplogroups; the CDY1a+ DAZ1/2 genes were deleted in 96.9% (217/224) of haplogroup D individuals, whereas the O lineage had a variety of gr/gr deletion types. The loss of CDY1a+ DAZ1/2 was not associated with spermatogenic impairment in haplogroup D (P = 0.33). CONCLUSIONS Taken together, gr/gr deletions in haplogroup D occur constitutively, are associated with the loss of CDY1a + DAZ1/2 and are phenotypically neutral. Further studies are needed to establish whether Y-linked compensatory factors outside the AZFc region can counteract the pathogenic effect of a gr/gr deletion in the D lineage.

Fresh testicular sperm retrieved from men with spinal cord injury retains equal fecundity to that from men with obstructive azoospermia via intracytoplasmic sperm injection

OBJECTIVE To examine the outcomes of intracytoplasmic sperm injection (ICSI) with testicular sperm retrieved from men with spinal cord injury. DESIGN Retrospective study. SETTING Private hospital-based infertility research laboratory. PATIENT(S) Twenty-two couples of whom one partner was a man with spinal cord injury (SCI). INTERVENTION(S) Reviewing the outcomes of testicular sperm extraction (TESE)-ICSI. MAIN OUTCOME MEASURE(S) Testicular sperm retrieval rate, fertilization rate, pregnancy rate, comparison with patients with obstructive azoospermia. RESULT(S) Testicular sperm were retrieved from 19 of 22 (86%) patients with SCI. Intracytoplasmic sperm injection resulted in a fertilization rate of 236 of 364 (65%). Of 19 couples, 14 couples achieved 18 pregnancies, and 22 infants (14 singleton and 4 twin) were born. (Pregnancy per couple was 74% and that per ICSI was 54%). There was no significant difference in pregnancy rate at the first ICSI between SCI couples and obstructive azoospermia couples (68% SCI, 68% obstructive azoospermia). However, pregnancy rate per fresh testicular sperm-ICSI was significantly higher than that per frozen-thawed sperm-ICSI in SCI couples (64% SCI fresh, 25% SCI frozen-thawed) although no significant difference was seen in obstructive azoospermia couples (76% obstructive azoospermia fresh, 63% obstructive azoospermia frozen-thawed). There was no significant difference in pregnancy rate between fresh ET cycle and frozen-thawed ET cycle in SCI couples. CONCLUSION(S) Testicular sperm in men with SCI may possess disadvantages in freezing and thawing compared with that in men with obstructive azoospermia. Fresh testicular sperm-ICSI may offer optimum outcome for SCI couples desirous of pregnancy.

Appearance of an oocyte activation-related substance during spermatogenesis in mice and humans

BACKGROUND Recently we reported that an oocyte activation ability in human and mouse sperm is associated with head flatness or the presence of perinuclear theca (PT) substance, MN13, which is an oocyte activation-related protein localized on the post-acrosomal sheath (PAS). As such, we hypothesize that the appearance of oocyte activation ability is stage-specifically regulated and depends on the formation of the acrosome or PAS/PT in spermatids. METHODS We monitored the appearance and movement of MN13 as a PT-specific molecule during spermatogenesis and analysed how the MN13 localization is affected in mouse and human globozoospermic acrosomeless sperm. RESULTS MN13 was first detected on the surface of acrosomic vesicles, i.e. on the nascent outer acrosomal membrane of step 5-6 round spermatids (Sb1 spermatids in human), and it was then translocated via the outer acrosomal membrane surface to the most distal region of the acrosome in step 7 round spermatids (Sb2 spermatids). As spermatids elongated, MN13 was translocated via the cytoplasmic space between the nuclear envelope and the overlying plasma membrane towards the post-acrosomal region, and it was organized on the top of the nascent PAS that was typically found in step 14 elongated spermatids (Sd1 spermatids). In contrast, MN13 was not found in any GOPC-deficient spermatids, which completely lack the acrosome but have manchettes (microtubule bundles), nor in mouse and human acrosomeless sperm. CONCLUSIONS The MN13 appearance or the MN13-related PAS/PT formation is highly dependant on acrosome formation; the MN13-related oocyte activation factor/ability is stage-specifically acquired in elongating/elongated spermatids.

Birth of a healthy infant after intracytoplasmic sperm injection using pentoxifylline-activated sperm from a patient with Kartagener’s syndrome

OBJECTIVE To describe the delivery of a healthy female infant after intracytoplasmic sperm injection (ICSI) using pentoxifylline-activated sperm from a patient with Kartageners syndrome. DESIGN Case report. SETTING Private assisted reproductive technology clinic in Japan. PATIENT(S) A couple with male factor infertility due to Kartageners syndrome. INTERVENTION(S) Intracytoplasmic sperm injection using ejaculated sperm activated by pentoxifylline. MAIN OUTCOME MEASURE(S) Semen characteristics, sperm ultrastructure, fertilization, pregnancy, and birth after ICSI. RESULT(S) The fertilization rate was 7 of 12 (58.3%), and the blastocyst formation rate was 4 of 7 (57.1%); all blastocysts were vitrified. After a single blastcyst transfer, a pregnancy ensued and progressed to term; a healthy female infant was delivered. CONCLUSION(S) With ejaculated sperm, which was activated by pentoxifylline, successful fertilization was accomplished by ICSI; thus, fertilization, vitrification, pregnancy, and delivery are attainable with sperm obtained from men with Kartageners syndrome.

Oocyte activation ability correlates with head flatness and presence of perinuclear theca substance in human and mouse sperm

BACKGROUND Recent studies indicate that round-headed sperm cannot activate oocytes and lack the postacrosomal sheath (PAS) or perinuclear theca (PT), although normal flat-headed sperm can activate oocytes and do have PAS (PT). In this study, we investigated how oocyte activation ability correlates with sperm head morphology (round and flat) and the presence of PT, by studying MN13, a representative molecule of the PT. METHODS We analyzed sperm with flat and round heads from infertile patients with globozoospermia (n = 1) and teratozoospermia (n = 1), and also from GOPC(-/-) mice, an animal model of human globozoospermia. Differential interference contrast image analysis, immunocytochemistry with MN13 antibody, transmission electron microscopy and an oocyte activation assay (assessing pronucleus formation) with ICSI were used. RESULTS Flat-headed (control) sperm from both a healthy fertile volunteer man and wild-type mice had MN13 and PAS (PT). Flat-headed sperm (<5% of the population) from GOPC(-/-) mice also had both MN13 and PAS (PT), and they showed high oocyte activation ability. In contrast, round-headed sperm from a globozoospermia patient (100%) and GOPC(-/-) mice (>95% of the population) had neither MN13, nor PAS (PT), nor oocyte activation ability. Oocyte activation was higher in flat- versus round-headed sperm from GOPC(-/-) mice (P < 0.05). CONCLUSIONS Oocyte activation ability may be related to sperm head flatness and presence of MN13 and PAS (PT) in human and mouse sperm. This information is a first step towards the possibility of selecting good-quality sperm with high oocyte activation ability for ICSI.

Physical and mental development of children after in vitro fertilization and embryo transfer

ObjectiveTo evaluate the physical and mental development of children after in vitro fertilization (IVF) and frozen embryo transfer (FET).MethodsBetween July 1995 and November 2003, 506 patients delivered 658 babies after IVF and ET treatment at our clinic (intracytoplasmic sperm injection (ICSI), 418; conventional IVF, (C-IVF) 125; FET, 115). A survey of the physical and mental developmental of the children was conducted by mailing questionnaires to parents. Comparisons were made between three treatment procedures, and development of singleton, twin and triplet delivery.ResultsThe response rate was 73.4% (483/658) for 324 children born after ICSI, 78 born after C-IVF, and 81 born after FET. The height and weight of assisted reproductive technology (ART) children at birth were significantly lower than that of naturally conceived babies (ART children: natural delivery; 46.8 cm, 49.0 cm and 2524 g, 3040 g, respectively). However, there was no significant difference between the singletons alone and naturally conceived children irrespective of the ART method. In addition, mental development was the same between singletons and naturally conceived children. The ART group tended to delay body development such as ‘holding their head uprs, ‘sitting up’, ‘crawl’ to moving growth in multiple births.ConclusionThe physical and mental development of twins or triplets was significantly more delayed than that of naturally conceived babies, but had improved to a similar extent of the singletons after the age of 6 months.