Human luteal cells have been reported to express human leukocyte antigen-DR and lymphocyte functional antigen-3 on the cell surface, suggesting physiological interaction between luteal cells and T-lymphocytes through the menstrual cycle into early pregnancy. To elucidate the role of peripheral lymphocytes on corpus luteum differentiation, the effect of peripheral blood mononuclear cells (PBMC) on steroidogenesis by luteal cells was investigated. The production of Th-2 cytokines such as interleukin (IL)-4 and IL-10 by the co-cultured cells was also examined, and the effects of these cytokines on progesterone production by luteal cells were investigated. Corpora lutea were obtained from eight non-pregnant women in the luteal phase and five women in early pregnancy for luteal cell culture. PBMC were isolated from unrelated women in the follicular phase, secretory phase, and early pregnancy. After coculture with allogenic PBMC for 48 h, progesterone production was significantly enhanced by PBMC from the secretory phase and early pregnancy in the non-pregnant luteal cell culture. In the pregnant luteal cell culture, a significant increase in progesterone production was also observed by the co-culture with PBMC from women in early pregnancy, showing that PBMC have a luteotrophic effect. The stimulatory effects of PBMC were also observed in co-culture conditions which prevented direct cell-to-cell interaction with luteal cells, showing the minor influence of mixed lymphocyte reaction. By co-culture with PBMC, the production of IL-10, but not IL-4, was significantly augmented in luteal cell culture derived from non-pregnant women, whereas the production of both IL-4 and IL-10 was significantly enhanced in the luteal cell culture derived from pregnant women. Moreover, IL-4 and IL-10 promoted progesterone production by cultured luteal cells, especially in the luteal cell culture derived from corpora lutea of early pregnancy. These findings indicate that PBMC stimulate progesterone production by luteal cells and suggest the involvement of PBMC in corpus luteum function and differentiation probably via the Th-2-type lymphocytes.

Background Serious complications continue to occur in laparoscopic cholecystectomy (LC). The commonly used indicators of surgical difficulty such as the duration of surgery are insufficient because they are surgeon and institution dependent. We aimed to identify appropriate indicators of surgical difficulty during LC. Methods A total of 26 Japanese expert LC surgeons discussed using the nominal group technique (NGT) to generate a list of intraoperative findings that contribute to surgical difficulty. Thereafter, a survey was circulated to 61 experts in Japan, Korea, and Taiwan. The questionnaire addressed LC experience, surgical strategy, and perceptions of 30 intraoperative findings listed by the NGT. Results The response rate of the survey was 100%. There was a statistically significant difference among nations regarding the duration of surgery and adoption rate of safety measures and recognition of landmarks. The criteria for conversion to an open or subtotal cholecystectomy were at the discretion of each surgeon. In contrast, perceptions of the impact of 30 intraoperative findings on surgical difficulty (categorized by factors related to inflammation and additional findings of the gallbladder and other intra-abdominal factors) were consistent among surgeons. Conclusions Intraoperative findings are objective and considered to be appropriate indicators of surgical difficulty during LC.

Preovulatory oocytes were collected from ovaries of beagle bitches that had received superovulatory treatment. They were cultured in a modified Krebs-Ringer bicarbonate solution containing 10% fetal calf serum and 30 mg/L gentamicin sulfate for up to 72 h. About 32% of oocytes reached metaphase II by 72 h of culture. When these in vitro-matured oocytes were inseminated with ejaculated beagle spermatozoa that had been preincubated for 4 h, sperm penetration of the zona pellucida started about 1 h after insemination, and both male and female pronuclei were seen in the ooplasm at 8 h after insemination. At 18-20 h after insemination, oocytes were transferred to Whitten's medium and cultured for 76-78 h. The first cleavage was observed at 48 h after insemination, and 15 of 45 oocytes developed to the 2-8-cell stage. These results demonstrate that in vitro-matured canine oocytes can be fertilized and develop to the 8-cell stage in vitro.

Background We previously identified 25 intraoperative findings during laparoscopic cholecystectomy (LC) as potential indicators of surgical difficulty per nominal group technique. This study aimed to build a consensus among expert LC surgeons on the impact of each item on surgical difficulty. Methods Surgeons from Japan, Korea, and Taiwan (n = 554) participated in a Delphi process and graded the 25 items on a seven-stage scale (range, 0-6). Consensus was defined as (1) the interquartile range (IQR) of overall responses ≤2 and (2) ≥66% of the responses concentrated within a median AE 1 after stratification by workplace and LC experience level. Results Response rates for the first and the second-round Delphi were 92.6% and 90.3%, respectively. Final consensus was reached for all the 25 items. 'Diffuse scarring in the Calot's triangle area' in the 'Factors related to inflammation of the gallbladder' category had the strongest impact on surgical difficulty (median, 5; IQR, 1). Surgeons agreed that the surgical difficulty increases as more fibrotic change and scarring develop. The median point for each item was set as the difficulty score. Conclusions A Delphi consensus was reached among expert LC surgeons on the impact of intraoperative findings on surgical difficulty.

Previously, it has been shown that integrin alpha6beta1 expressed on human granulosa cells regulates luteinization in co-operation with its ligand, laminin. In this study, integrin alpha2 was immunohistochemically demonstrated to be expressed on granulosa and large luteal cells. It was also detected on luteinizing theca interna cells after ovulation. Immunoreactive collagen type IV, which is one of the ligands for integrin alpha2beta1, was detected around granulosa cells in the pre-ovulatory follicles and its expression was rapidly increased during ovulation. By flow cytometry, collagen type IV was detected on the cell surface of luteinizing granulosa cells isolated from pre-ovulatory follicles, confirming the physiological interaction between granulosa cells and collagen type IV. Collagen type IV in follicular fluid was positively related with progesterone concentration. In 4-day cultures of granulosa cells, collagen type IV in the media was significantly increased by human chorionic gonadotrophin (HCG). The progesterone production was significantly attenuated when granulosa cells were cultured on collagen type IV-coated dishes, suggesting that collagen type IV suppresses granulosa cell luteinization. These findings show that collagen type IV, a ligand for integrin alpha2beta1, is rapidly produced around luteinizing granulosa cells during ovulation, probably under the control of luteinizing hormone (LH) and suggest that collagen type IV is a new parameter and/or regulator of granulosa cell luteinization in the periovulatory phases.