M. Nagayoshi

An investigation of the potential effect of vacuoles in human sperm on DNA damage using a chromosome assay and the TUNEL assay

BACKGROUND The aims of this study were to establish whether individual differences exist in the frequency and size of vacuoles found in human sperm and to ascertain whether such vacuoles are involved in causing DNA damage. METHODS Morphologically normal sperm were obtained from 15 IVF and 2 ICSI patients and 3 fertile donors. (i) Sperm heads were analyzed for the presence of vacuoles under a 1000× differential interference contrast microscope. (ii) In three patients and two donor samples, structural chromosomal damage was evaluated in normal sperm containing large vacuoles and selected at 1000× magnification for injection into mouse oocytes. (iii) In 10 patients and two donor samples, confocal laser microscopy detected DNA damage in sperm-exhibiting large vacuoles and stained with an in situ cell death detection kit. RESULTS (i) Vacuoles were observed in almost all normal sperm from patient and donor ejaculates and were mainly located at the tip or middle area of the sperm heads. However, average incidence of normal sperm exhibiting large vacuoles was 4.6 and 4.2% in the patient and donor groups, respectively. (ii) Sperm chromosome assays did not reveal any differences in the incidence of structural chromosome aberrations between sperm exhibiting large vacuoles and those without them (9.1 versus 4.1%). (iii) No significant difference in frequency of TUNEL-positive cells was found between normal sperm with large vacuoles and those without them in the samples examined. Among 227 sperm exhibiting large vacuoles, only 7 cells were TUNEL positive. CONCLUSION The results showed that large vacuoles were not responsible for DNA damage, suggesting that intra-cytoplasmic injection of morphologically selected sperm may not be required for patients who produce high-quality semen.

Human sperm head vacuoles are physiological structures formed during the sperm development and maturation process

OBJECTIVE To clarify whether human sperm vacuoles affected intracytoplasmic sperm injection (ICSI) success rates. DESIGN Retrospective study. SETTING A private infertility clinic. PATIENT(S) Spermatozoa and spermatids were obtained from 11 normozoospermic, 10 oligozoospermic or asthenozoospermic, 4 obstructive azoospermic, and 3 nonobstructive azoospermic men. INTERVENTION(S) Differential interference contrast observation and intracytoplasmic injection of morphologically selected sperm. MAIN OUTCOME MEASURE(S) Incidence, size, and position of vacuoles of sperm cells were recorded. Ability of fertilization and blastocyst development were compared between cells with and without vacuoles. RESULT(S) More than 97.4% of ejaculated, 87.5% of epididymal, 87.5% of testicular spermatozoa, and more than 90.0% of Sc-Sd2 spermatids had vacuoles of various sizes. The incidence of vacuoles on ejaculated cells was significantly higher than that on the other types of cells, but there was no difference between sperm from normozoospermic men and those from the other donors. Removal of plasma membrane and/or acrosome did not affect the incidence of vacuoles. Although more than 60% of spermatozoa had small vacuoles in the acrosomal regions, 52.6% of Sb1-2 spermatids had large vacuoles. After injection of a motile spermatozoon with large and small vacuoles, 60.9% and 85.7% of metaphase II oocytes could be normally fertilized, respectively, and almost half of the zygotes developed to the blastocyst stage. When using sperm without vacuoles, the fertilization rate was 80.0%, but only 25% of them developed to the blastocyst stage. CONCLUSION(S) Human sperm head vacuoles did not affect ICSI outcomes.

Fourteen babies born after round spermatid injection into human oocytes

Significance Men without spermatozoa or elongating spermatids in their testes have been considered sterile and are advised to consider using a sperm donor. However, these men may have round spermatids. We have been able to accurately identify these cells based on their structural and physical characteristics (verified by karyotyping and FISH). Round spermatid injection was effectively used in our clinic and resulted in the birth of 14 healthy babies. Although the current success rate of round spermatid injection is not very high compared with intracytoplasmic sperm injection, this procedure can be the last resort for men who cannot produce spermatozoa but wish to use their own genetic material to produce offspring. During the human in vitro fertilization procedure in the assisted reproductive technology, intracytoplasmic sperm injection is routinely used to inject a spermatozoon or a less mature elongating spermatid into the oocyte. In some infertile men, round spermatids (haploid male germ cells that have completed meiosis) are the most mature cells visible during testicular biopsy. The microsurgical injection of a round spermatid into an oocyte as a substitute is commonly referred to as round spermatid injection (ROSI). Currently, human ROSI is considered a very inefficient procedure and of no clinical value. Herein, we report the birth and development of 14 children born to 12 women following ROSI of 734 oocytes previously activated by an electric current. The round spermatids came from men who had been diagnosed as not having spermatozoa or elongated spermatids by andrologists at other hospitals after a first Micro-TESE. A key to our success was our ability to identify round spermatids accurately before oocyte injection. As of today, all children born after ROSI in our clinic are without any unusual physical, mental, or epigenetic problems. Thus, for men whose germ cells are unable to develop beyond the round spermatid stage, ROSI can, as a last resort, enable them to have their own genetic offspring.

Preimplantation Genetic Diagnosis and Natural Conception: A Comparison of Live Birth Rates in Patients with Recurrent Pregnancy Loss Associated with Translocation

Background Established causes of recurrent pregnancy loss (RPL) include antiphospholipid syndrome, uterine anomalies, parental chromosomal abnormalities, particularly translocations, and abnormal embryonic karyotypes. The number of centers performing preimplantation genetic diagnosis (PGD) for patients with translocations has steadily increased worldwide. The live birth rate with PGD was reported to be 27-54%. The live birth rate with natural conception was reported to be 37-63% on the first trial and 65-83% cumulatively. To date, however, there has been no cohort study comparing age and the number of previous miscarriages in matched patients undergoing or not undergoing PGD. Thus, we compared the live birth rate of patients with RPL associated with a translocation undergoing PGD with that of patients who chose natural conception. Methods and Findings After genetic counseling, 52 patients who desired natural conception and 37 patients who chose PGD were matched for age and number of previous miscarriages and these comprised the subjects of our study. PGD was performed by means of fluorescence in situ hybridization analysis. The live birth rates on the first PGD trial and the first natural pregnancy after ascertainment of the carrier status were 37.8% and 53.8%, respectively (odds ratio 0.52, 95% confidence interval 0.22-1.23). Cumulative live birth rates were 67.6% and 65.4%, respectively, in the groups undergoing and not undergoing PGD. The time required to become pregnancy was similar in both groups. PGD was found to reduce the miscarriage rate significantly. The prevalence of twin pregnancies was significantly higher in the PGD group. The cost of PGD was

Genetic risk of Klinefelter's syndrome in assisted reproductive technology

7,956 U.S. per patient. Conclusions While PGD significantly prevented further miscarriages, there was no difference in the live birth rate. Couples should be fully informed of the similarity in the live birth rate, the similarity in time to become pregnancy, the advantages of PGD, such as the reduction in the miscarriage rate, as well as its disadvantages, such as the higher cost, and the advantages of a natural pregnancy, such as the avoidance of IVF failure. The findings presented here should be incorporated into the genetic counseling of patients with RPL and carrying a translocation.

Are Crater Defects in Human Sperm Heads Physiological Changes during Spermiogenesis and Epididymal Maturation

The main cause of Klinefelters syndrome (KS) has been believed to be XY sperm. Accordingly, in the intracytoplasmic sperm injection treatment of patients with KS, hereditary KS has been a concern. Therefore, this study attempted to estimate the risk before and after the assisted reproductive technology.

Development of new procedure to reduce the amount of recipient cytoplasm (mitochondria) at the M-II karyoplast transfer for the treatment of mitochondrial diseases

Abstract We observed the crater defects (CD) of human sperm head at a magnification of×1,000 with an oil immersion lens and using an inverted microscope equipped with Nomarski optics and scanning and transmission electron microscope. Motile and normally shaped sperm with three different sizes of CD were selected and injected into M-II oocytes, donated by a consenting human patient and superovulated mice. On average, 1.54 CDs were confirmed in almost all (95.9%) sperm head, and the incidences of CDs did not change after the various demembraned pretreatments. The CDs were also found in epididymal and testicular sperm and elongating and elongated spermatids, but the incidences were a little fewer than ejaculated sperm. No CDs were found in mouse and boar sperm irrespective of the pretreatments. The fertilization and blastocyst formation rates were not significantly different following the ICSI using human sperm with 3 sizes of CDs. We speculate that CDs are not abnormal findings and physically structural changes during sperm maturation process.

Prediction of embryonic development and clinical outcome following ICSI according to the stages of nuclear maturation division

cleavage stage Finnish cycles (184.3 141.0 IU), compared with U.S. blastocyst cycles (low eSET use: 154.2 129.5, P<0.0001; high eSET use: 158.1 223.8, P<0.0001). The estimated number of cleavage stage transferrable embryos was higher in the U.S. blastocyst cycles (low eSET use: 9.6 5.5; high eSET use: 8.8 5.4) than in cleavage stage Finnish cycles (5.8 3.3, P<0.0001). CONCLUSIONS: Single blastocyst transfer in the U.S. is associated with higher ctLBR than cleavage stage embryo transfer in Finland. Probable reasons for these findings are high selectivity of patients for blastocyst eSET in the U.S. which constitute only 3.7% of all fresh cycles. These women have better ovarian response, as indicated by lower use of gonadotropins during stimulation and more embryos available for transfer, compared to Finnish cleavage stage patients, who are selected for eSET on a much wider basis. Supported by: Helsinki University Hospital.

Preimplantation diagnosis of repeated miscarriage due to chromosomal translocations using metaphase chromosomes of a blastomere biopsied from 4- to 6-cell-stage embryos

fertilization rate was 80.0%, 57.1%, 76.2%, 50.0% respectively (overall mean 1⁄4 67.3%). In the control group the normal fertilization rate was 79.1%. The normal fertilization rate was significantly lower in oocytes which were cultured from MI to MII stage than in the control group (67.3% VS 79.1%; P < 0.05). The overall rate of blastocyst development in the MI to MII cultured group was 38.5% (5/13), and good quality blastocysts 30.8% (4/13). In the control group the rates were 32.1%(433/1349) and 26.0%(351/1349)respectively. There was no significant difference between these two groups. CONCLUSION: This work suggests that in cases where only a few oocytes or only MI oocytes are retrieved, additional culture and observation of PB release to time ICSI may be useful in improving embryo development potential. Future work should include further consideration of the optimum timing of PB release for ensuring the best embryo development potential.