Reduction of connexin 43 in human cumulus cells yields good embryo competence during ICSI.

PURPOSE: The purpose of this study was to predict developmental competence of human oocytes during ICSI via analysis of connexin 43 (Cx43) in cumulus cells surrounding mature oocytes. MATERIALS AND METHODS: Human cumulus cells were manually separated from the oocyte-cumulus complex under a microscope. Cx43 mRNA was expressed by real-time quantitative polymerase chain reaction (RT-PCR) measurement in cumulus cells. RESULTS: There was no significant relationship between expression of Cx43 and fertilisation or cleavage rate. However, Cx43 expression was lower in the good morphology group (blastomeres>7 cells with fragmentation<10% on day 3) when compared to the other groups (p=0.035). CONCLUSIONS: These results suggest that full reduction of Cx43 expression on cumulus cells at the time of oocyte collection during ICSI is essential for developmental competence of human oocytes.

Sperm retention site and its influence on pronucleus stage evaluation following intracytoplasmic sperm injection.

Aim: It has been suggested that the position of the sperm after intracytoplasmic sperm injection (ICSI) has an effect on the development and quality of the embryo. In this study, we retrospectively examined whether pronucleus stage evaluation used through clinical studies in recent years has relevance with regard to sperm location. Methods: From 2003 to 2005, 1285 oocytes from 459 patients (average age: 36 years) were retrospectively analyzed. The 459 patients underwent ICSI because of fertilization disorders and oligozoospermia. Follicle stimulation was via either Clomid or the long protocol. Human chorionic gonadotropin was administered to induce ovulation and oocyte retrieval was conducted 35 h later. After confirming the presence of a polar body, we immobilized the ovum at the 6 o'clock position, introduced the injection pipette at the 3 o'clock position and carried out ICSI. Results: When a sperm was located at a position that was opposite to the polar body, both classifications of Scott and Tesarik regarding embryo quality were distinctly low. Furthermore, a good embryo classification ensued when the sperm was located adjacent to the polar body. Conclusion: The zone in which the sperm was located did not always correlate with embryo quality; however, our study suggested that sperm location affects the synchronization of the nucleolus. When carrying out ICSI, it is important to take into consideration the insertion point of the sperm. (Reprod Med Biol 2007; 6: 171-174).

Sperm retention site and its influence on cleavage rate and early development following intracytoplasmic sperm injection.

BACKGROUND: Intracytoplasmic sperm injection (ICSI) has risen to the forefront of reproductive technology. In the present study, the location of the sperm injection was noted, and a prospective study was conducted to evaluate the effect of the sperm retention site on cleavage rates and embryo quality after ICSI. METHODS: This study involved 336 ICSI patients (age 27-44; average 37.4) where 1545 oocytes were observed. An oocyte was divided into nine sites and the sperm retention site was observed microscopically after injection. The polar body was placed at either the twelve or six o'clock position. The injection pipette was introduced at the three o'clock position and oolemma rupture was ascertained by mild suction. The main outcome measures were the relationship of sperm remaining in position in the oocyte to fertilization rate and embryo quality. RESULTS: When the injection pipette was introduced at the three o'clock position, about 80% of the sperm remained in the center or left of center. The fertilization rate was significantly lower (p < 0.05) when the sperm remained near the site of introduction. Embryo quality was not significantly affected by the sperm retention site. CONCLUSIONS: About 12-14% of the spermatozoa remained near the introducing position, and in these cases the fertilization rate was low. However, once fertilization occurred, the sperm retention site had minimal impact on embryo quality. Injecting sperm near the spindle site may improve embryo quality.

Serum steroids concentration might be used for monitoring the growth of follicles in friendly in vitro fertilization.

Background: Steroid levels have been used as the predictive parameters for oocyte maturation and embryo development. In the present study, estradiol and progesterone concentrations in the follicular fluid and serum were evaluated in conventional in vitro fertilization (IVF; follicle stimulating hormone [FSH] and/or human menopausal gonadotropin [hMG] after pituitary desensitization) and friendly IVF (no stimulation, clomiphene citrate, small dose of FSH or hMG without pituitary desensitization). The purpose of the present study was to evaluate the differences in steroid distribution between conventional and friendly IVF. Methods: Concentrations of estradiol, progesterone, FSH, and luteinizing hormone (LH) in serum and follicular fluid were determined in conventional and friendly IVF protocols by an enzyme-linked immunosorbent assay kit. Correlations between follicular fluid and serum steroid concentrations in these different protocols, and between pregnant cycles and steroid concentrations were evaluated. Results: Two hundred and thirty-four samples of follicular fluid from 74 IVF patients were analyzed. In conventional IVF, there was no relationship in steroid levels in between follicular fluid and serum steroids, whereas serum steroid concentrations correlated with the number of developing follicles. There was a relationship between the serum and follicular fluid estradiol levels (r = 0.467, P < 0.0001) as well as progesterone levels (r = 0.227, P = 0.0488) from friendly IVF patients. Conclusions: Serum steroid concentrations were mainly associated with the number of developing follicles. In the cases of friendly IVF, which had a small number of developing follicles, serum steroids might be used to monitor follicular fluid steroid concentrations. (Reprod Med Biol 2006; 5: 277-282).

Reduction of progesterone receptor expression in human cumulus cells at the time of oocyte collection during IVF is associated with good embryo quality.

BACKGROUND: It has been reported that the progesterone receptor (PR) level is transiently increased within the follicle by LH stimulation and controls cumulus cells in follicles and oocyte maturation. The purpose of this study was to predict developmental competence of human oocytes during IVF via analysis of PR in cumulus cells surrounding mature oocytes. METHODS: Prior to oocyte retrieval, the follicular diameter was measured and follicular fluid was collected from each mature follicle. Cumulus cells were manually separated from the oocyte-cumulus complex under a microscope. PR and PR mRNA were assessed by immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR) measurement in human cumulus cells. RESULTS: Immunoreactive PR-A was mainly localized in the cytoplasm and PR-B was localized in the nuclei. There was no significant relationship between PR expression and follicular diameter, follicular fluid concentration of steroids, or LH. There was no significant relationship between expression of PRs and fertilization or cleavage rate. However, PR expression was lower in the good morphology group (blastomeres > or =7 cells with fragmentation > or =5% on day 3) when compared to the other groups (P<0.05). CONCLUSIONS: These results suggest that follicular LH or steroids do not affect PR expression, and full reduction of total PR expression on cumulus cells at the time of oocyte collection is associated with good morphology in human oocytes.

Preventive effect of recombinant human lactoferrin on lipopolysaccharide-induced preterm delivery in mice.

BACKGROUND: In order to investigate whether recombinant human lactoferrin (rh-LF) has the same effect as bovine LF (b-LF) for the prevention of preterm delivery, we conducted the following animal studies. METHODS: Female C3H/HeNCrj mice were pair-mated with male Crj:B6D2F1 mice. As a model of preterm delivery, on day 15 of gestation, a 50 microg/kg intraperitoneal injection of lipopolysaccharide (LPS) was administered twice with a 3-hr interval between injections (14:00 and 17:00 hours). At 1 hr prior to each LPS injection (13:00 and 16:00 hours), an intraperitoneal injection of saline, b-LF, or rh-LF (1 mg/body) was administered. In non-LPS-treated controls, an intraperitoneal injection of saline was administered four times (13:00, 14:00, 16:00, and 17:00 hours). We measured body weight and recorded delivery time. To measure plasma levels of interleukin-6 (IL-6), other pregnant mice, in which the same preparation as mentioned above had been done, were killed 6 h after the second LPS injection and blood samples were obtained. RESULTS: Delivery occurred in preterm (16.2 +/- 0.4 days of gestation) in all LPS-treated mice not administered LF. LF significantly prolonged gestation of LPS-treated mice: LPS + b-LF, 17.8 +/- 0.3 days; LPS + rh-LF, 18.2 +/- 1.3 days (p < 0.05). LF (1 mg/body) significantly suppressed plasma IL-6 in LPS-treated mice: LPS + b-LF, 1060 +/- 154; LPS + rh-LF, 244.2 +/- 59.4; and LPS without LF, 1628 +/- 115 pg/ml (p < 0.05). CONCLUSIONS: rh-LF has an effect of prolongation of gestation in LPS-induced preterm delivery in mice, suppressing LPS-induced plasma IL-6 augmentation.

Correlations between steroids concentration in follicular fluid, pronuclear morphology and embryo qualities in in vitro fertilization.

Background: Several parameters of early embryo development are known as predictors of implantation success. Recently, zygote or embryo morphological assessments are thought to be a major method of selection in embryo transfer. We expected that the concentrations of the steroids in follicular fluid (FF) were associated with oocyte maturation and embryo quality. In the present paper, we evaluated the relationship of several parameters. Methods: We investigated 105 samples of FF from 22 subjects by in vitro fertilization (IVF). We evaluated the correlations between the FF concentrations of estradiol (E2) and progesterone (P4), the diameter of the ovarian follicles, fertilization, and zygote assessment based on pronuclear morphology and day 3 embryo qualities (i.e. number of blastomeres and fragmentation rate). Results: There was a positive correlation between the E2 concentrations in FF and serum (r = 0.273, P < 0.01), but there was no correlation between follicular diameter and the FF concentration of each steroid. The concentration of E2 in FF containing fertilized oocytes was not significantly different from that in FF containing unfertilized oocytes. At the pronuclear stage, the concentration of either steroid in FF did not differ among the morphological groups. The concentration of P4 in FF was significantly lower in the group in which pronuclei were detected at 20 h after insemination than in the group in which pronuclei were not detected. The concentration of E2 in FF was significantly related to the number of blastomeres (r = 0.271, P < 0.05) and furthermore, was significantly higher in FF from which morphologically good embryos were obtained at day 3 (P < 0.05). Conclusions: The FF concentrations of the steroids did not affect the pronuclear pattern, but P4 production may play a role in reducing the potential of the oocyte to develop pronuclei and the concentration of E2 may predict the cleavage capability of the oocyte. (Reprod Med Biol 2003; 2: 171-176).