Metabolic profiling of human follicular fluid identifies potential biomarkers of oocyte developmental competence.

The use of metabolomic based techniques to aid oocyte and embryo selection has gained attention in recent years. Previous work from our laboratory has demonstrated that the (1)H NMR-based metabolic profile of follicular fluid correlates with oocyte developmental potential. Patients undergoing IVF at the Merrion Fertility Clinic had follicular fluid collected at the time of oocyte retrieval. The fatty acid composition of follicular fluid from follicles where oocytes fertilised and developed into multi-cell embryos (n=15) and from oocytes that fertilised normally but failed to cleave (n=9) (cleaved vs non-cleaved) was compared. Statistical analysis was performed on the data using univariate and multivariate techniques. Analysis of the fatty acid composition revealed that there were nine fatty acids significantly different between follicular fluid from the cleaved and the non-cleaved sample groups. Of particular interest were the higher concentration of total saturated (P=0.03) and the lower concentration of total polyunsaturated fatty acids in the non-cleaved sample group (P=0.001). Random forest classification models were used to predict successful cleavage in follicular fluid samples producing models with errors rates of <10%. Receiver operating characteristic analysis demonstrated that the model had good predictability with an area under the curve of 0.96. The panel of fatty acid biomarkers identified in this study indicates that the fatty acid composition of follicular fluid may be more predictive in comparison to other previously identified biomarkers. Following validation in a larger cohort, these biomarkers may have the potential to be used in fertility clinics to aid the selection of oocytes in the future.

Viral screening of couples undergoing partner donation in assisted reproduction with regard to EU Directives 2004/23/EC, 2006/17/EC and 2006/86/EC: what is the evidence for repeated screening?

BACKGROUND: This paper concerns the requirements of the EU Tissue and Cells Directives with regard to the biological screening of donors of reproductive cells which are to be used for partner donation. METHODS: We review the evidence regarding the risks of transmission of blood-borne viruses [hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency virus (HIV)] in the assisted reproductive technology (ART) setting. We document the experience in seven Irish ART clinics since the introduction of the legislation. RESULTS: Even among those known to be HBV-, HCV- or HIV-positive, when current best practice ART procedures are employed for gamete and embryo processing, cross-contamination in the ART facility or horizontal or vertical transmission to a partner or neonate has never been documented. When samples are processed and high-security straws are used for cryopreservation, transmission of virus and cross-contamination in storage have not been reported. CONCLUSIONS: While initial screening of those about to embark on ART treatment is good practice, we can find no medical or scientific evidence to support re-screening prior to each treatment cycle for individuals undergoing partner donation in ART. It would seem more appropriate to focus on risk reduction using a combination of initial baseline screening (with a reduced frequency of re-testing), appropriate sample processing and best possible containment systems for cryostorage.

Reducing twin pregnancy rates after IVF--elective single embryo transfer (eSET).

Multiple pregnancy is a major complication of IVF and is associated with increased maternal, fetal and neonatal morbidity. Elective single embryo transfer (eSET) during IVF, rather than the more standard transfer of two embryos (double embryo transfer or DET), has been shown to significantly reduce the multiple pregnancy rate associated with IVF, while maintaining acceptable pregnancy rates. Couples undergoing IVF in 2008 who met good prognostic criteria had eSET performed. Pregnancy and twinning rates were compared with those for similar couples in 2007 who had DET. Couples unsuccessful with a fresh cycle of treatment had subsequent frozen embryo transfer cycles with DET. The cumulative pregnancy rate was similar for each group. However there were no multiple pregnancies in the eSET group, compared to 4 twins of 5 pregnancies in the DET group. 96% of eligible couples agreed to eSET. ESET is successful in and acceptable to good prognosis Irish couples undergoing IVF.

Frozen-thawed transfer cycles: are they comparable with fresh?

Cryopreservation of zygotes and subsequent thaw and transfer is an established ART treatment. We assessed if success rates frozen-thawed (day 2) zygotes are comparable with the outcome in fresh cycles of treatment. We performed a prospective follow-up and analysis of all frozen (FZT) and fresh cycles of treatment during a 12 months period. One hundred and nineteen patients in the frozen-thawed and 652 in the fresh group had a transfer. The overall thaw-survival rate was 71.7%. Clinical pregnancy rates per thaw and transfer were respectively 15.1% and 21% in the frozen and 29.1% (per transfer) in the fresh group. Implantation rates in fresh and frozen cycles were 16% and 12.3% respectively. The pregnancy loss rate was higher in the FZT group (29% vs. 18.3%). Cryopreservation of good quality zygotes, after fresh transfer offers optimal success rates in subsequent frozen treatment. It also encourages consideration of elective single zygote transfers.

A randomized prospective study of early follicular or midluteal initiation of long protocol gonadotropin-releasing hormone in an in vitro fertilization program.

OBJECTIVE: To determine the optimum menstrual cycle time to initiate a long-protocol gonadotropin-releasing hormone agonist (GnRH-a) down-regulation regimen before hMG stimulation before IVF. DESIGN: Randomized, prospective, single, first cycle study. SETTING: University teaching hospital. PATIENTS: Eighty-six infertile couples undergoing IVF-ET attempt under rules for Ireland. INTERVENTION: Gonadotropin-releasing hormone agonist administered intranasally from day 1 or 21 of menstrual cycle. Human menopausal gonadotropin commenced when pituitary down-regulation was confirmed. MAIN OUTCOME MEASURES: Ovarian response, cancellation, fertilization, and pregnancy rates. RESULTS: No significant differences found between day 1 and day 21 initiation. But starting on day 1 is more easily recognizable by patients and avoids the possibility of administering GnRH-a in the presence of an unsuspected pregnancy. CONCLUSIONS: Both follicular and luteal phase initiation of GnRH-a long-protocol down-regulation are equally efficacious. In our clinical context, patients and management favor commencing on day 1.

Microbial contamination in an in vitro fertilization-embryo transfer system.

OBJECTIVE: To determine sources and transmission of microorganisms in IVF-ET and efficacy of in-place controlling systems. DESIGN: Prospective study. SETTING: In Vitro Fertilization-Embryo Transfer Unit at a university teaching hospital. PATIENTS: Twenty-eight couples undergoing 30 completed IVF-ET cycles. INTERVENTIONS: Gamete and embryo processing in a penicillin and streptomycin-rich medium. MAIN OUTCOME MEASURES: Presence of microorganisms at various stages of IVF-ET. Fertilization, cleavage, and pregnancy rates. RESULTS: In 50% of cycles no microorganisms were isolated and in the other 50% microbes were cultured from various loci. Cultures of four preprocessed semen samples were positive and corresponding postprocessed samples negative. Microbes were detected in 27% of needle washes after oocyte collection; in 40% and 32% of follicular fluids from left and right ovaries, respectively; and in two culture media from egg-sperm incubations at 20 hours after insemination. No microorganisms were grown from media from zygote incubations. Fertilization, cleavage, and pregnancy rates were independent of microbial presence. CONCLUSION: Seminal fluid and transvaginally collected oocytes are potential sources of microbial contamination of the IVF-ET culture system. A penicillin- and streptomycin-rich culture medium is effective in removing contaminating microbes. End point measures are not affected by commensal contamination.

Processing of semen in an antibiotic-rich culture medium to minimize microbial presence during in vitro fertilization.

OBJECTIVE: To identify and determine the prevalence of microorganisms in preprocessed and postprocessed semen in an IVF-ET program. DESIGN: Prospective study. SETTING: University Teaching Hospital. PATIENT(S): Seventy-four men undergoing preprogram evaluation, each producing two semen samples. INTERVENTION(S): Semen processing with a wash and swim-up technique in a penicillin- and streptomycin-rich medium. MAIN OUTCOME MEASURE(S): The identity and prevalence of seminal microorganisms before and after processing. RESULT(S): Sixty-three percent of individual unprocessed semen samples grew microorganisms, the majority of which were nonpathogenic. Thirty-three men (44.6%) had microbes identified in repeat samples, four had identical organisms each time. Twenty (27%) had positive cultures in one sample, negative in the other. Twenty-one (28.4%) had consistently sterile semen. After seminal processing, the recovery rate for microbes was 5%. Microbial presence after processing did not correlate with either the sperm swim-up concentration or the initial microbial concentration. CONCLUSION(S): Bacteriospermia is common. The microorganisms found rarely are replicated and most likely represent contamination. Wash and swim-up semen preparation in an antibiotic rich culture medium effectively eliminates 95% of organisms.

Are seminal fluid microorganisms of significance or merely contaminants?

OBJECTIVE: To determine the contribution of urethral and skin flora to seminal fluid cultures and the relation between bacteriospermia and seminal leukocytes. DESIGN: Prospective study. SETTING: IVF-ET unit at a university teaching hospital. PATIENT(S): Sixty men starting an IVF-ET program. INTERVENTION(S): Culture of sequential first-catch urine, midstream urine, and semen samples with evaluation of seminal leukocytes. MAIN OUTCOME MEASURE(S): A comparison of microbes from first-catch urine, midstream urine, and semen samples and the correlations of seminal microbes, elevated leukocyte concentrations, and pregnancy. RESULT(S): Microorganisms were detected in 37% of first-catch urine samples, 27% of midstream urine samples, and 51% of semen samples. Most microorganisms were gram-positive microbes and were common to both urine and semen samples. Mean and median leukocyte concentrations were 0.98 x 10(6)/mL and 0.10 x 10(6)/mL, respectively. There was no correlation between seminal microbes and raised leukocytes or between leukocytospermia and/or bacteriospermia and pregnancy. CONCLUSION(S): Microorganisms are commonly found in insignificant quantities in the semen of asymptomatic men. The frequent isolation of gram-positive microbes common to both urine and semen and the absence of a correlation with raised leukocyte concentrations suggest that bacteriospermia most commonly represents contamination.

Should gonadotropin-releasing hormone down-regulation therapy be routine in in vitro fertilization?

OBJECTIVE: To compare the classic clomiphene citrate (CC) and hMG regime for ovarian stimulation before IVF in women who received hMG post-long protocol down-regulation with either 3 mg triptorelin [INN] IM or 150 mg buserelin acetate four times daily intranasally. Furthermore, if possible, to determine the preferred method of down-regulation. DESIGN: A prospective study of 150 women randomized blind to the clinician to one of three alternative ovarian stimulation regimes when passing for the first time through an IVF program during 1992. RESULTS: Triptorelin [INN] down-regulated significantly more quickly than buserelin acetate. The non-down-regulated group CC and hMG used significantly less hMG in a shorter time. In these women LH levels at hCG administration were significantly higher. No other intergroup differences were found. Pregnancy and take-home baby rates for the overall study were, respectively, 32%:25% (per cycle) and 42%:33%; (per ET) for the triptorelin [INN] group 28%:22% and 39%:31%; the CC group 32%:24% and 46%:34%; and the buserelin acetate group 34%:28% and 42%:34%. CONCLUSIONS: Triptorelin [INN] and buserelin acetate were comparable in all parameters except down-regulation. The former was significantly quicker and more sure. In none of the clinical end points measured, however, was the classic CC and hMG non-down-regulation regime significantly less effective or troublesome than where down-regulation was used. These results therefore show that although indications for down-regulation before IVF exist, it should not be used on all patients.

Mid to late luteal phase steroids in minimal stage endometriosis and unexplained infertility.

Serum and peritoneal fluid concentrations of progesterone and 17 beta-oestradiol were measured during days 20-27 of the luteal phase in 33 women with minimal stage endometriosis and 21 with unexplained infertility. The results were analysed using Student's t-test and straight line regression analysis. In both groups, 17 beta-oestradiol and progesterone values were several-fold higher than the corresponding serum values and there were significant correlations between the peritoneal fluid and the serum steroid values. In the peritoneal fluid there was a significant reduction in both progesterone and its ratio with 17 beta-oestradiol in women with minimal stage endometriosis (P < 0.03, P < 0.04, respectively). The peritoneal fluid levels of 17 beta-oestradiol in women with minimal stage endometriosis were, however, not significantly altered. Despite the correlations between peritoneal fluid and serum steroid levels, there were no significant changes in the serum concentrations of progesterone, 17 beta-oestradiol or the ratio of progesterone to 17 beta-oestradiol in women with minimal stage endometriosis. It is concluded that luteal phase progesterone and its relationship to 17 beta-oestradiol are subtly altered in infertile women with minimal stage endometriosis compared with women with unexplained infertility. These alterations may be more clearly demonstrated in peritoneal fluid rather than in peripheral blood, possibly because the latter contains steroids which are of non-ovarian origin and are also constantly affected by the liver enzyme activity and enterohepatic circulation of individual subjects.

The value of subcellular elemental analysis in the assessment of human spermatozoa.

The conventional sperm parameters of concentration, motility and morphology are descriptive and have proved of little value in predicting the fertilization potential of semen. New complementary tests of sperm function are required. Previous studies evaluating the subcellular elemental composition of spermatozoa using X-ray microanalysis with electron microscopy suggested that this technique may have the potential to confirm spermatozoal intracellular normality. This study compares the semen from 18 men of subfertile relationships and from 10 men of proven fertility with reference to concentration, motility, morphology and the elemental composition of spermatozoa. The elements phosphorus, sodium, potassium, chlorine and sulphur were detected and there were large intra- and intersample variations in the concentrations of these elements. There was no statistically significant difference in the elemental composition between the two groups. This study suggests that the spermatozoa of fertile and subfertile men are similar in subcellular elemental composition and in the maintenance of an ionic gradient. This test is therefore not a useful predictor of sperm function.

Preparation of antibody free spermatozoa by in vitro immunodepletion using immunobeads.

In the present study ejaculates from six infertile men with antisperm antibodies were processed on a six step discontinuous percoll gradient. This treatment yielded a sperm fraction with a different percentage of sperm with antisperm antibodies. The resultant sperm preparation was incubated with immunobeads. Sperm were recovered following incubation by swim-up. Sperm recovered at the end of processing showed reduced populations of antisperm antibodies. Even though sufficient numbers of sperm were obtained to consider assisted reproduction, reduced motility was recorded in four of the ejaculates when compared with routine processing. Following trial preparations of semen samples this technique may play a role in sperm selection for assisted conception.

A mid-luteal phase comparison of peritoneal fluid volume and its content of PGF2 alpha and PGE2 in women with minimal stage endometriosis and a normal pelvis.

The study was carried out following the approval of our Hospital Ethics Committee and an informed patient consent. Samples of peritoneal fluid were collected from the Pouch of Douglas at laparoscopy from 32 subjects with minimal stage endometriosis (MSE) and 16 without any visible signs of endometriosis (normal group). All the subjects were in 20 to 23 day period of their menstrual cycle which was histologically confirmed. The endometriosis score was estimated according to the American Fertility Score, 1985 and the fluid samples were collected in tubes containing heparin/indomethacin and standing in ice. The samples were estimated for volume, erythrocyte count, prostaglandin(PG) F2 alpha, and PGE2. Radioimmunoassay techniques were used for the estimation of PGs. Prostaglandin F2 alpha, was measured in its parent form while PGE2 was converted into bicyclic-PGE2 before estimation. The results indicate that the peritoneal fluid in women with MSE has higher than normal levels of PGF2 alpha and lower than normal levels of PGE2 and its PGE2: PGF2 alpha ratio is also below normal. There is no difference in the volume of peritoneal fluid in the two groups. Provided the fluid sample is not contaminated with abdominal blood its erythrocyte count is not related to its prostaglandin content.

Does patient semen quality alter during an in vitro fertilization (IVF) program in a manner that is clinically significant when specific counseling is in operation?

PURPOSE: It has been shown that the stress of infertility may impair semen quality. Whether counseling can attenuate this affect is unproven. This study examines, in an IVF program setting, where specific counseling is in operation, whether semen collected on the day of oocyte recovery is significantly different from that obtained during the prior clinical suitability assessment of the couple. RESULTS: In the 125 consecutive couples examined, there were no significant overall differences in semen volume or sperm density. There was a significant increase in sperm motility on the day of oocyte retrieval (P < 0.001). Twenty-three patients (18.4%) showed an increase in quality, and 21 (16.8%) a decrease, on the day of oocyte recovery. The environment of production appeared to exert no influence. Fertilization failure occurred in seven couples, three (14%) of whom presented for the first time with decreased semen parameters on the day of IVF. This compares with 1 of 23 (4%) fertilization failure in those whose parameters increased. CONCLUSION: The semen quality of the vast majority of the patients studied in this highly counseled program does not appear to be significantly affected by the superstress of participation in the day of oocyte recovery.