To determine if oxysterol levels, produced by macrophages, are elevated in peritoneal fluid of women with endometriosis. Prospective Case-Control. Forty women (age 18-40 years) undergoing scheduled laparoscopy (diagnostic or bilateral tubal ligation) were consented and participated in the study. 25 participants were diagnosed with endometriosis. 15 participants had no evidence of endometriosis (controls). One to 10 mL of peritoneal fluid was aspirated from the posterior cul-de-sac from each participant. The fluid was evaluated to determine the role of oxysterols in endometriosis. In addition, human endometrial stromal cells (HESC) were treated with either synthetic 22-hydroxycholesterol (22-HC), 25-HC and 27-HC or peritoneal fluid samples from study participants to determine levels of steroidogenic acute regulatory (StAR) protein, StAR mRNA and pregnenolone synthesis. Among the 25 study participants with endometriosis, 3 had Stage I (minimal), 16 had Stage II (mild), 2 had Stage III (moderate) and 4 had Stage IV (severe) endometriosis per American Society for Reproductive Medicine scale for classification of endometriosis. Preliminary data indicates that levels of 25-HC and 27-HC increased in a stage-dependent manner in the peritoneal fluid of women with endometriosis. In addition, StAR protein and mRNA expression were strikingly higher in peritoneal fluid from participants with Stage IV endometriosis compared with controls. HESC treated with either peritoneal fluid from study participants or with oxysterols showed increases in StAR protein, StAR mRNA expression and pregnenolone levels. Oxysterols, oxygenated metabolites of cholesterol secreted by macrophages, are elevated stage dependently in peritoneal fluid of women with endometriosis. HESC treated with peritoneal fluid samples showed steroidogenic activity by increased StAR protein, StAR mRNA expression and pregnenolone synthesis. Detection of either 25-HC or 27-HC levels through blood or peritoneal fluid sampling may lead to less invasive and less costly techniques to diagnose endometriosis or to determine its recurrence. Additionally, pharmaceutical advances directed at inhibiting the production of these oxysterols by macrophages may lead to new treatment options for patients with endometriosis.

Polycystic ovary syndrome (PCOS) is associated with clinical and laboratory hyperandrogenism. Hyperandrogenism is exacerbated by hyperinsulinemia. Foods containing high levels of starch and dairy induce an exacerbated insulinotropic response. The purpose of the study was to determine the effect of an 8-week low starch/low dairy diet on clinical and laboratory evidence of hyperandrogenism. Prospective cohort including women with PCOS ages 18 to 45 years. Twenty-one participants with PCOS were enrolled. 18 completed the study (3 did not complete the study due to noncompliance). Participants received 1 hour of dietary instruction from a registered dietician at the beginning of the study. Participants were permitted to eat ad libitum lean meats, non-starchy vegetables, nuts and low sugar fruits to the point of satiety. Participants returned in 8 weeks for repeat testing. Testosterone and free testosterone assays were run by one laboratory (Quest Diagnostics Nichols Institute, San Juan Capistrano, CA). Ferriman-Gallwey scores were performed by one physician at the beginning and end of the 8-week dietary intervention. Following an 8-week dietary modification, change in total and free testosterone levels were not normally distributed (p=.049 and p=.013 respectively). Both total testosterone levels (median change= -7.0, IQR=31.0ng/dL; p=.065) and free testosterone levels decreased (median change= -0.25pg/mL, IQR=4.4pg/mL; p=.200) though the change was not statistically significant. However, modified Ferriman-Gallwey hirsutism scores were reduced (median change= -2.0, IQR=5.0; p=.018) despite only 8-weeks of intervention. Reduction in modified Ferriman-Gallwey hirsutism scores were achieved after a brief 8-week dietary modification. Further studies are needed to determine if dietary intervention may be successful in lowering total and free testosterone levels in women with PCOS.

It is well documented that sperm are susceptible to shock during processing which often induces biochemical pathways leading to cell death. Previous animal studies from this lab suggest a modification of the collection technique (Device for Improved Semen Collection; DISC) can prevent activation of these pathways, yielding larger, more motile pools of sperm for treatment and resulting in higher pregnancy rates. The objective of the present study was to evaluate the DISC in humans. Lab-based trials in donors and infertility patients. Donors collected in a standard specimen cup (SSC) and the DISC. The samples were then processed and cultured for 24 h. Aliquots were taken, over time, for semen analysis using CASA, and biochemical assessment including: acrosomal status, lipid peroxidation, mitochondrial membrane potential (MMP) and DNA damage. A preliminary clinical trial was then conducted comparing the DISC to SSC. Couples undergoing IUI alternated semen collections between the DISC and SSC for up to 6 cycles. Donor samples collected in the DISC exhibited improved semen parameters when compared to the SSC: viability (P<.005), motility rates (P=.066), path velocities (P=.061), progressive velocity (P<.05), and motility index (P<.034). Further, cells collected in the DISC had more intact acrosomes (P<.017), and retained higher MMP (P<.004). Twenty-four couples completed 51 IUI cycles (26 SSC vs. 25 DISC). As in the donor trial, samples from the DISC trended to have higher motility (P=.063) and progressive velocity (P=.057). There were 9 pregnancies (17.6%) with equivalent results in the DISC vs. SSC (4 vs. 5; P=.762). However, while 100% of the pregnancies in the DISC delivered, only 40% in the SSC (P=.058) did. As in the animal studies, results suggest improved semen quality from the DISC. Larger numbers are needed to determine if improved semen quality will lead to the increased pregnancy rates seen in other species.

This review presents the methodology of using theoretic models for development of cryopreservation protocols by designing specific cooling profiles and selecting appropriate external conditions to optimize cryopreservation survival. Biophysical events during the processes of cryopreservation were examined and corresponding theoretic equations were used to simulate cryopreservation procedures under various slow cooling conditions for rat zygotes in the presence of DMSO, using a 0.25-mL plastic straw as the container. Simulation revealed three regions with their own characteristics and cryopreservation relevance. In addition, this review discusses vitrification cryopreservation using two-step additions. The effects of exposure durations and exposure temperatures on cell survival and subsequent development rates were examined in a series of cryopreservation experiments. Values of accumulative osmotic damage were used to quantitatively examine the magnitude of the associated osmotic damage during cryoprotective agent (CPA) additions and dilutions. In these investigations, oocyte blastocyst rates were highly correlated with the values of accumulative osmotic damage in the processes of CPA additions/dilutions. This review emphasizes the most essential step of the selection of the cell container in the process of cryopreservation, and provides practical suggestions and guidelines for optimizing slow cooling protocols. The review stresses that conducting CPA addition steps at 25 °C would be preferable for vitrification. It also suggests that the final dilution process needs more systematic research to optimize vitrification procedures.

OBJECTIVE:Previous research has suggested a correlation between the subtle rise in progesterone induced by the administration of human chorionic gonadotropin (hCG) and cycle outcome in patients undergoing in vitro fertilization with embryo transfer (IVF-ET). The data suggest that patients who have <2-fold increase in progesterone levels will not conceive as a result of that IVF-ET attempt (poor responders). Further, if the luteinizing-granulosa cells of poor responders are placed in culture, their steroid hormone production will be significantly less than that of the luteinizing-granulosa cells of patients with a normal response to hCG (>3-fold; normal responders). Recent studies have demonstrated that steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroid hormone biosynthesis. The objective of the present study was to determine whether StAR expression differed in normal responders and poor responders undergoing IVF-ET. STUDY DESIGN:The luteinizing-granulosa cells of 6 patients were isolated after follicular aspiration and assayed for StAR expression. Three sets of cells were from patients exhibiting a normal response to hCG administration and 3 sets were from patients exhibiting a poor response. RESULTS:Data suggest a 17% drop in StAR protein in patients with a poor response to hCG administration when compared with those with a normal response. CONCLUSIONS:Although StAR protein was expressed in all 6 cellular extracts, expression appeared greatest in cells recovered from normal responders. Further, although two thirds of the patients with a normal response reported term pregnancies, no pregnancies were reported in the poor responders. Low levels of progesterone and absence of pregnancy may be due to a defect in the mechanism that converts granulosa and theca-lutein cells to luteal cells after the luteinizing hormone surge (or hCG administration). Data from the current study would suggest that the StAR protein may be involved.

Objectives: Previous research from this and other laboratories has suggested a correlation between the subtle rise in Progesterone (P) induced by the administration of hCG, and cycle outcome in patients undergoing in vitro fertilization with embryo transfer (IVF-FT). The data suggests patients who have <2-fold increase in P following hCG will not conceive on that IVF-ET attempt (Poor responders; PR). Further, if the luteinizing-granulosa cells (LGC) of PR are placed in culture, their steroid hormone production will be significantly less than the LGC of patients with a normal response to hCG (>3-fold; NR). Recent studies have demonstrated steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroid hormone biosynthesis. The objective of the present study was to determine if StAR expression differed in NR and PR patients undergoing IVF-ET. Design: A comparison of StAR protein expression from the LGC of IVF-ET patients with a normal (N=3) or poor (N=3) rise in progesterone following the administration of hCG. Materials and Methods: The LGC of six women undergoing treatment of infertility by IVF-ET were isolated following follicular aspiration, and snap frozen in liquid nitrogen. Previous P levels measured at 12 h before and 12 h after hCG administration had demonstrated three of the patients exhibited a NR to hCG administration and three a PR. To verify the expression of StAR, whole-cell extracts were subjected to Western blot analysis using standard techniques. Results: As designed, patients with a poor response to hCG administration had a significantly smaller increase in P (1.76-fold ± .23) when compared to the patients with a normal response (3.65 fold ± .77). While Western blot analysis demonstrated a band correlating with StAR in all six cell extracts, the bands from NR cells appeared much denser than those from PR cells in side-by-side runs. These data await confirmation after standardization for total cellular proteins. Similar to previous studies, pregnancy was reported in 2/3 of the patients with a NR to hCG administration. However, none of the PR group reported a pregnancy. Conclusions: There is increasing evidence that the subtle rise in progesterone seen prior to ovulation is necessary for the establishment of pregnancy. Previous work has demonstrated that a subpopulation of women undergoing IVF-ET for the treatment of infertility do not exhibit this rise in P, possible due to a defect in the mechanism which converts granuloma and theca to luteal cells after the LH surge (or hCG administration). Data from the current study would suggest that the StAR protein, which regulates steroidogenesis, might be involved. This project was supported by an Institutional Seed Grant from Texas Tech University Health Sciences Center.