Clinical outcomes of transfer of frozen and thawed single blastocysts derived from nonpronuclear and monopronuclear zygotes.

PURPOSE: In assisted reproductive technology, normal zygotes are bipronuclear (2PN) during fertilization confirmation; however, sometimes, nonpronuclear zygotes (0PN) and monopronuclear zygotes (1PN) are found during routine observations. METHODS: To elucidate the clinical usefulness of in vitro-fertilized embryos, we investigated the rates of clinical pregnancy, live birth, miscarriage, and congenital abnormality after transfer of frozen-thawed 1PN- and 0PN-derived single blastocysts at Denentoshi Ladies Clinic, Kanagawa, Japan. RESULTS: The rates of pregnancy and live birth for 1PN-derived blastocysts obtained by conventional in vitro fertilization were 37.5% and 27.1%, respectively, which was not significantly different from those for 2PN-derived blastocysts; however, the rates for 0PN-derived blastocysts were significantly lower. The pregnancy and live birth rates for 0PN-derived embryos obtained by intracytoplasmic sperm injection (ICSI) were 45.7% and 34.8%, respectively, which was not significantly different from those for 2PN-derived blastocysts; however, the rates for 1PN-derived blastocysts were significantly lower (4.0% for both) than those for 2PN- and 0PN-derived blastocysts. No congenital abnormalities were found in infants resulting from transfer of 0PN- or 1PN-derived blastocysts. CONCLUSIONS: Both 1PN- and 0PN-derived blastocysts can be used for embryo transfer; however, care should be taken in making decisions about 1PN-derived blastocysts, especially if they are obtained by ICSI.

Identification of placental leucine aminopeptidase and triton-slowed aminopeptidase N in serum of pregnant women.

BACKGROUND: Previously, we found characteristic triton-slowed bands of aminopeptidase N (APN) in cholestatic serum by triton-polyacrylamide gel electrophoresis (triton-PAGE) [Makoto Kawai, Yukichi Hara, Triton-polyacrylamide gel electrophoresis and leucine aminopeptidase activity staining detect Triton-slowed bands including high-molecular-mass aminopeptidase N (CD13) isoform in cholestatic patient sera. Clin Chim Acta 2006; 364:188-195]. METHODS: Sera from 14 normal pregnant women were electrophoresed on polyacrylamide gel containing 0.02 l/l triton (triton-PAGE) or a 0-0.02 l/l horizontal gradient of triton (gradient-triton-PAGE), and stained with leucine-beta-naphthylamide. Some samples were pretreated with a monoclonal APN antibody or rabbit anti-placental leucine aminopeptidase (PLAP) serum. The stained bands were eluted from the gel, treated with N- and O-glycosidase, and analyzed by Western blotting with rabbit anti-APN or anti-PLAP serum. RESULTS: Triton-PAGE clearly differentiated 5 LAP activity bands (1-5 from the front). Gradient-triton-PAGE revealed that bands 4-5 were slowed by triton (triton-slowed bands) much more than bands 1-3. Triton-PAGE of antibody-treated serum showed that bands 1, 2, 4, and 5 are mainly APN and that band 3 is PLAP. The molecular mass of PLAP was about 130-140 kDa before treatment with glycosidases but 100 kDa after. Triton-PAGE detected PLAP in 13 and triton-slowed APN in 4 of the 14 women. CONCLUSIONS: Triton-PAGE differentiates PLAP from APN. Triton-slowed APN as well as PLAP is present in the serum of pregnant women.

Methylation of the calcium channel-related gene, CACNA2D3, is frequent and a poor prognostic factor in gastric cancer.

BACKGROUND & AIMS: The calcium channel voltage-dependent alpha2delta subunit consists of 4 genes, CACNA2D1 to CACNA2D4, of which CACNA2D2 and CACNA2D3 are located on 3p21.3 and 3p21.1, respectively. Here, we examined the relation between alpha2delta subunit gene alterations and gastric carcinogenesis. METHODS: The expression and methylation status of the alpha2delta subunit genes were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and methylation-specific PCR in gastric cancers (GCs). The effects of CACNA2D3 expression were examined by cell proliferation and adhesion assays, and they predicted target gene alterations. RESULTS: Aberrant methylation of CACNA2D1 and CACNA2D3 mostly corresponded to their expression status in GC cell lines. CACNA2D1/3 methylation was detected in 10 (12.5%) and 24 (30%) of the 80 GC cases, respectively, but no CACNA2D2 methylation was seen in 32 cases. CACNA2D3 methylation was more frequently found in diffuse type than in intestinal type (16/38 [42.1%] vs 8/42 [19.0%]; P = .025) GCs. Among the 53 patients with advanced GCs, patients with cancers showing CACNA2D3 methylation had a significantly shorter survival time than patients without this methylation (P = .003). Exogenous CACNA2D3 expression strongly inhibited cell growth and adhesion and up-regulated p21 and p27 expression in HEK-293T and NUGC4 cells. Inverse effects were seen by CACNA2D3 small interfering RNA treatment in the CACNA2D3-positive cell lines, indicating that CACNA2D3 may have tumor suppressive functions. CONCLUSIONS: Loss of CACNA2D3 expression through aberrant promoter hypermethylation may contribute to gastric carcinogenesis, and CACNA2D3 methylation is a useful prognostic marker for patients with advanced GC.

Increase in the concentration of cytosolic-free calcium induced by human follicular fluid was decreased in single human spermatozoon with abnormal morphology.

Aim: The increase in the concentration of cytosolic-free calcium ([Ca2+]i) induced by follicular fluid or progesterone has been reported to promote an acrosome reaction and alternation in several motion parameters in human sperm (hyperactivation). We previously reported that populations of sperm in cell suspension obtained from infertile men with abnormal morphology exhibited lower mean peak progesterone-evoked [Ca2+]i compared with morphologically normal sperm using cell-suspension methods. In the present study, the change in [Ca2+]i in individual normally and abnormally shaped spermatozoa was compared. Methods: The change in [Ca2+]i induced by human follicular fluid in individual spermatozoa with normal and abnormal morphology was compared using the fluorescent calcium-sensitive dye fluo-3/AM. The spatial distribution of the increase in [Ca2+]i in single sperm was also investigated. Results: The [Ca2+]i of normally shaped spermatozoa increased rapidly after the administration of human follicular fluid. The response reached a peak within 2-3 s and then slowly declined to a plateau phase. The baseline and peak fluorescence in spermatozoa with abnormal morphology was lower when compared with normal spermatozoa. The follicular-fluid-induced increase in [Ca2+]i (expressed as a percentage increase in [Ca2+]i over basal) in morphologically abnormal sperm was 39.2 ± 5.3% (n = 107, mean ± standard error), which was smaller than that of morphologically normal sperm (61.6 ± 5.7%, n = 100, P < 0.005) from seven healthy donors. The follicular-fluid-induced [Ca2+]i increases observed in sperm with morphologically abnormal mid-pieces (20.9 ± 4.3%, n = 12, P < 0.05) or tails (40.7 ± 6.0%, n = 92, P < 0.05) were lower than those of morphologically normal spermatozoa (61.6 ± 5.3%, n = 101). The follicular-fluid-induced [Ca2+]i increase of morphologically normal spermatozoa from infertile couples (35.1 ± 6.3%, n = 25, P < 0.05) was also found to be lower than that of morphologically normal spermatozoa from healthy donors. Conclusion: The present study shows that spermatozoa with abnormal morphology in healthy donors have disorders of signal transduction, as do normally shaped sperm in men from infertile couples. (Reprod Med Biol 2008; 7: 143-149).

Impaired hyperactivation of human sperm in patients with infertility.

Hyperactivation and acrosome reaction are prerequisite steps for sperm to be able to fertilize an oocyte. In mammals, hyperactivation is defined as a movement pattern seen in spermatozoa at the site and time of fertilization. The objectives of the present experiments were to analyze the process of hyperactivation and to investigate its relationship with progesterone evoked intracellular calcium concentration ([Ca2+]i) increase and their implications with infertility. After capacitation the sperm from patients, when compared with donor's sperm, showed decreased percentage of hyperactivated sperm, molitily, progressive motility, and curvilinear velocity (VCL). On the other hand, the linearity (LIN) was increased. Amplitude of lateral head displacement (ALH) and [Ca2+]i increase (peak and plateau from baseline) showed good correlation in patients with infertility. These data suggest that impaired hyperactivation might be involved in the pathophysiology of infertility.

Ca(v)2.3 (alpha1E) Ca2+ channel participates in the control of sperm function.

To know the function of the Ca2+ channel containing alpha(1)2.3 (alpha1E) subunit (Ca(v)2.3 channel) in spermatozoa, we analyzed Ca2+ transients and sperm motility using a mouse strain lacking Ca(v)2.3 channel. The averaged rising rates of Ca2+ transients induced by alpha-D-mannose-bovine serum albumin in the head region of Ca(v)2.3-/- sperm were significantly lower than those of Ca(v)2.3+/+ sperm. A computer-assisted sperm motility assay revealed that straight-line velocity and linearity were greater in Ca(v)2.3-/- sperm than those in Ca(v)2.3+/+ sperm. These results suggest that the Ca(v)2.3 channel plays some roles in Ca2+ transients and the control of flagellar movement.