Influence of male human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection on the reproductive outcomes in serodiscordant couples: a case-control study.

BACKGROUND: Nowadays, serodiscordant couples (SDCs) with human immunodeficiency virus (HIV) or hepatitis C virus (HCV)-infected men have the chance to conceive safely, giving birth with a minimum risk of cross-infection. OBJECTIVE: To assess the impact of male HIV and HCV infection on the assisted reproductive technologies (ART) outcomes in SDCs, with HIV or HCV seropositive men and negative partners. MATERIALS AND METHODS: Of 153 couples: 24 in Group 1 (HIV-seropositive men), 60 in Group 2 (HCV-seropositive men) and 69 in Group 3 (controls). Sperm-washing procedure was performed using a three-step system. Fresh ICSI cycles were carried out in HIV SDCs, HCV SDCs and controls. Seminal parameters, fertilization rate (FR), cleavage rate (CR), pregnancy rate per cycle (PR/C), miscarriage rate, implantation rate (IR) and live birth rate were evaluated. RESULTS: All the seropositive men have undetectable viral loads at the time of insemination, and both partners were free from co-morbid infections. The median number of embryos transferred was 2.0 (IQR 1.0-3.0), with no differences among groups. FR was significantly reduced in HIV and HCV SDCs compared to the controls (66%, 61% and 75%, respectively; p < 0.01). CR was similar between groups (p = 0.3). IR was 12.1%, 11.1% and 14.1%, respectively, in the three groups (p = 0.30). PR/C was 21.7%, 17.6% and 20.2% in HIV, HCV and controls, respectively. Live birth rate per cycle was 17.4%, 15.7% and 15.9%, respectively. There were no significant differences in clinical pregnancies per cycle, as well as miscarriages and live births (p = 0.30; 0.30; 0.60, respectively). CONCLUSIONS: The sperm-washing technique with ICSI may generate a promising way to improve pregnancy outcomes and to reduce the risk of viral transmission in these couples. In this setting, we can correctly counsel HIV- and HCV-infected men of SDCs with regard to the likelihood of father their own biological child.

Are hormone measurements and ultrasounds really predictors of sperm retrieval in testicular sperm extraction? A case report and literature review.

Azoospermia can be diagnosed in about 10%-15% of the infertile male population. To overcome the problem of failure to produce spermatozoa in the ejaculate in patients with nonobstructive azoospermia (NOA), testicular sperm extraction (TESE) may be performed to find the focal area of spermatogenesis. A 47-year-old man with NOA presented for treatment of secondary couple infertility. The patient underwent a first TESE 7 years earlier with cryopreservation, and an intracytoplasmic sperm injection-embryo transfer ended in a term pregnancy. He reported a history of repeated testicular traumas. At the present time, a complete medical workup was carried out, including clinical history, general and genital physical examination, scrotal and transrectal ultrasounds. Hormone measurements showed follicle-stimulating hormone level of 42.7 IU/L, luteinising hormone of 11.4 IU/L, total testosterone of 2.6 ng/ml and right and left testicular volume, respectively, of 4 and 3.9 ml. He underwent a second TESE, with successful sperm retrieval and cryopreservation. The histological pattern was hypospermatogenesis. In cases of extreme testicular impairment, although in the presence of very high follicle-stimulating hormone value and small testicular volume, estimating poor sperm recovery potential, the integration of clinical and anamnestic data, could help the surgeon to practise the more appropriate method of treatment.

Hormonal patterns, steroid receptors and morphological pictures of endometrium in hyperstimulated IVF cycles.

OBJECTIVE: The purpose of this contribution is to investigate the pathophysiology of the abnormal endometrial development in hyperstimulated IVF cycles. STUDY DESIGN: In 12 IVF-patients who did not have embryo transfer because of failure of oocyte fertilization, serum values of 17 beta-estradiol, progesterone, FSH, LH, total and free testosterone, and androstenedione were measured on the pick-up day and were evaluated with respect to the values normally expressed in the day of ovulation; in the endometrial specimens collected 2 days later, at the time of embryo replacement, estrogen and progesterone receptors were immunohistochemically determined and dating by the Noyes method was performed. RESULTS: 17 beta-Estradiol values are constantly higher, and progesterone levels are, only in four cases, higher than expected for the day of ovulation in a natural cycle. These hormonal patterns can only partially explain the pattern of steroid receptors: progesterone receptors are expressed sparsely both in glands and stroma, while estrogen receptors are abundant in the glands and absent in the stroma. In 11 of 12 patients an abnormal endometrial development with stromal advancement was observed: this morphological picture of the endometrium could partially be explained only in the four cases presenting high progesterone levels by serum values and endometrial receptor content of estrogen and progesterone. CONCLUSIONS: The abnormal endometrial development in hyperstimulated IVF cycles could only in part be explained by estrogen and progesterone, and other factors have to be considered.

Two functional assays of sperm responsiveness to progesterone and their predictive values in in-vitro fertilization.

We have recently reported, in a small cohort of subjects, that acrosome reaction (AR) and intracellular free calcium ([Ca2+]i) increase in response to progesterone were significantly correlated with in-vitro fertilization (IVF) rate. In the present study we extended these results to 90 subjects undergoing IVF. We confirm that both parameters were highly significantly correlated with the fertilization rate (P < 0.001). In particular, significantly lower responses to progesterone were detected in subjects with a fertilization rate < 50%, further enlightening the functional significance of sperm responsiveness to progesterone with respect to the process of fertilization. Moreover, we report here that both tests are highly discriminant of fertilization success, with positive predictive values > 90% for [Ca2+]i values which increase by > 1.2-fold and AR inducibility > 7% (cut-off values). Conversely, AR following challenge with the calcium ionophore A23187 was less significantly correlated with the percentage fertilization rate (P < 0.05), and showed lower predictive values than response to progesterone. All these tests ([Ca2+]i increase in response to progesterone, AR in response to progesterone and to A23187) appear highly sensitive and moderately specific. The positive predictive value may rise to > 95% when the combination of two tests ([Ca2+]i and inducibility of AR in response to progesterone) is considered. No correlation with fertilization rate has been found for spontaneous AR or basal [Ca2+]i. In conclusion, we propose that assessment of human sperm responsiveness to progesterone may be clinically useful in predicting fertilizing ability in vitro.

Intracellular calcium increase and acrosome reaction in response to progesterone in human spermatozoa are correlated with in-vitro fertilization.

In this study we have investigated responsiveness to progesterone in spermatozoa from a group of unselected male partners of couples undergoing in-vitro fertilization (IVF). We evaluated progesterone-stimulated intracellular Ca2+ ([Ca2+]i) and percentage increase in acrosome reaction in the same sperm sample used for oocyte inseminations. [Ca2+]i was measured with a fluorimetric method, while the acrosome reaction was assessed using a fluorescent probe (fluorescein isothiocyanate-labelled peanut lectin). The average percentage [Ca2+]i as well as the rate of increase in the frequency of acrosome reaction following progesterone challenge were significantly lower (P < 0.005) in the group of patients with a fertilization rate < 50%. In addition, significant correlations between the fertilization rate and the progesterone-stimulated [Ca2+]i and acrosome reaction increases (r = 0.78 and r = 0.79 respectively) were observed. Furthermore, in cases of fertilization failure, no increase of [Ca2+]i or acrosome reaction was observed in response to progesterone with the exception of one case. Our results indicate that [Ca2+]i and acrosome reaction increases in response to progesterone can be of value in the prediction of sperm fertilizing ability. As the two parameters were significantly correlated to each other (r = 0.86), the two assays have similar IVF predictive value and might be used interchangeably as a diagnostic tool in the assignment of male patients to the different kinds of assisted fertilization techniques.

Estrone 3-glucuronide chemiluminescence immunoassay (LIA) and 17beta estradiol radioimmunoassay (RIA) in the monitoring of superovulation for in vitro fertilization (IVF): correlation with follicular parameters and oocyte maturity.

Many works in the literature of the last years had reported that urinary approach to superovulation study is a suitable method to evaluate ovarian response to pharmacological stimulation. Before applying urinary determination of hormonal levels with a chemiluminescence immuno assay (LIA) method in early morning urine (EMU) samples, we had studied the correlation of RIA-LIA procedures with reference to follicular volumes at hCG day and to recovered oocyte maturity; in fact follicular growth and oocyte morphological features are the main parameters to evaluate a successful induced cycle. In our department the IVF cycles are daily monitored with RIA seric E2 and LIA E1-3G determination, besides ultrasound examination of follicular growth. We have studied E2 and E1-3G levels on the hCG administration day and their correlation with follicular areas and volumes; moreover, we have evaluated hormonal values on oocyte pick-up day with reference to recovered oocyte number and maturity. We have assumed as good timing for oocyte pick-up when more than 50% of recovered oocytes were of good quality (maturity score 4). We have observed that the highest pre ovulatory E1-3G value is consistent with the best timing for oocyte pick-up; it's possible to obtain a conversion coefficient follicular volumes and urinary E1-3G excretion. We have not found significant differences between plasmatic and urinary estrogenic parameters. It is important to remember the advantages connected by a not isotopic and not invasive method. The absence of discomfort for the patients may be a decisive factor to choose the monitoring method and LIA procedure may represent a valid alternative to RIA.

Effects of 5-methyltetrahydrofolate on the activity of fluoropyrimidines against human leukemia (CCRF-CEM) cells.

The growth inhibitory effects of 5-fluorouracil (FUra) or 5-fluoro-2'-deoxyuridine (FdUrd) combined with 5-methyltetrahydrofolate (5-CH3-H4PteGlu) were determined, as a function of time, dose, and sequence of exposure, on human T-lymphoblast leukemia cells, CCRF-CEM. Synergistic inhibitory effects on cell growth were obtained when exponentially growing CCRF-CEM cells were exposed to 5-CH3-H4PteGlu (1-100 microM) for 4 hr and to FUra (250 microM) or FdUrd (0.5 microM) during the last 2 hr. Synergism was dependent on 5-CH3-H4PteGlu dose (100 greater than 10 greater than 1 microM) and did not occur at 0.1 microM. No clear dependence of synergism on sequence was observed with FUra and 5-CH3-H4PteGlu combinations (5-CH3-H4PteGlu----FUra,5-CH3-H4PteGlu + FUra, or FUra----5-CH3-H4PteGlu). With 5-CH3-H4PteGlu and FdUrd combinations, synergism was dependent on sequence of exposure (5-CH3-H4PteGlu + FdUrd, 5-CH3-H4PteGlu----FdUrd were synergistic, but FdUrd----5-CH3-H4PteGlu was not). Thymidine (0.1 microM), added after drug treatment, substantially rescued CCRF-CEM cells from 5-CH3-H4PteGlu----FUra cytotoxicity. L-methionine (1500 mg/l) completely protected CCRF-CEM cells from enhanced cytotoxicity of the combination, 5-CH3-H4PteGlu-FdUrd. The results are consistent with the hypothesis that the mechanism by which 5-CH3-H4PteGlu potentiates fluoropyrimidine cytotoxicity is the enhancement of complex formation between thymidylate synthase and 5-fluorodeoxyuridylate, as a consequence of an increase of intracellular levels of 5,10-methylenetetrahydrofolate generated from 5-CH3-H4PteGlu. Also, enhanced stability of the complex in the presence of high levels of this folate coenzyme may contribute to the synergism observed. These data provide a rationale basis for further trials of folate coenzymes and fluoropyrimidine combinations in the clinic.

Enhancement of fluoropyrimidine cytotoxicity by 5-methyltetrahydrofolate in a human leukemia cell line, CCRF-CEM.

The inhibitory effects of combined 5-methyltetrahydrofolate (5-CH3-THF), the physiological circulating folate species, and fluoropyrimidines, 5-fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd), on growth of human leukemia cells, CCRF-CEM, were determined as a function of time, dose, and sequence of exposure. Exposure of CCRF-CEM cells in exponential growth to 5-CH3-THF (1-100 microM) for 4 h and to FUra (250 microM) or FdUrd (0.5 microM) during the last 2 h resulted in having synergistic inhibitory effects on cell growth. Synergy was dependent on 5-CH3-THF dose (100 greater than 10 greater than 1 microM) and did not occur at 0.1 microM. No clear dependency of synergy on sequence was observed with FUra and 5-CH3-THF combinations (4 h exposure, 5-CH3-THF----FUra, 5-CH3-THF + FUra, or FUra----5-CH3-THF). With 5-CH3-THF and FdUrd combinations, synergy was dependent on sequence of exposure (5-CH3-THF----FdUrd and 5-CH3-THF + FdUrd were synergistic, but FdUrd----5-CH3-THF was not). Thymidine (0.1 microM), added after drug treatment, substantially rescued CCRF-CEM cells from 5-CH3-THF-FUra cytotoxicity. L-Methionine (1500 mg/l) completely protected CCRF-CEM cells from the toxicity of the combination 5-CH3-THF-FdUrd. The results are consistent with the hypothesis that the mechanism by which 5-CH3-THF potentiated fluoropyrimidine cytotoxicity is the enhancement of ternary complex formation between thymidylate synthase and 5-fluorodeoxyuridylate, the active metabolite of fluoropyrimidines, as a consequence of an increase of intracellular levels of 5-10-methylenetetrahydrofolate generated from 5-CH3-THF.

Modulation of fluoropyrimidine cytotoxicity by methotrexate or 5-methyltetrahydrofolate in human leukemia cells in vitro.

The effects of methotrexate, 5-fluorouracil, 5-fluoro-2'-deoxyuridine on the growth of human leukemic T-lymphoblasts, CCRF-CEM, were determined as a function of drug concentration and exposure time. Substantial inhibition of cell growth (greater than or equal to 90%) was obtained with short duration of exposure (4 h) for MTX (ED90 = 4.3 microM). 5-fluorouracil was a relatively ineffective cytotoxic agent for exposure of short duration (4 h). Only exposure of 24 and 72 h resulted in cell growth inhibition greater than or equal to 90% with this drug. In terms of a ED90, 5-fluoro-2'-deoxyuridine was about 190- and 1300-fold more active than 5-fluorouracil for 24 and 72 h exposures, respectively (0.4 vs 75 microM and 0.01 vs 26 microM). Sequential exposure to methotrexate (4 h) and 5-fluorouracil during the last 2 h of methotrexate exposure resulted in synergistic inhibitory effects on cell growth. Antagonistic inhibitory effects on cell growth of methotrexate and 5-fluoro-2'-deoxyuridine combinations were observed independently of drug concentrations. Pretreatment (4h) with 5-methyltetrahydrofolate, the reduced folate to which leucovorin is rapidly converted in vivo, potentiated cell growth inhibitory effects of subsequently administered 5-fluorouracil or 5-fluoro-2'-deoxyuridine. These results provide information on scheduling of methotrexate or reduced folates and fluoropyrimidines that might have potential importance in the development of clinical trials designed for patients with leukemia and lymphoma.