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.

High variability in results of semen analysis in andrology laboratories in Tuscany (Italy): the experience of an external quality control (EQC) programme.

We report the results of the first three trials of an external quality control (EQC) programme performed in 71 laboratories executing semen analysis in Tuscany Region (Italy). At the end of the second trial, participants were invited to attend a teaching course illustrating and inviting to adhere to procedures recommended by WHO (V edition). Results of the first three trials of the EQC documented a huge variability in the procedures and the results. The highest variability was found for morphology (CV above 80% for all the trials), followed by count (CV of about 60% for all the trials) and motility (CV below 30% for all the trials). When results of sperm count and morphology were divided according to the used method, mean CV values did not show significant differences. CV for morphology dropped significantly at the third trial for most methods, indicating the usefulness of the teaching course for morphology assessment. Conversely, no differences were observed after the course for motility and for most methods to evaluate count, although CV values were lower at the second and third trial for the laboratories using the Burker cytometer. When results were divided according to tertiles of activity, the lowest mean bias values (difference between each laboratory result and the median value of the results) for count and morphology were observed for laboratories in the third tertile (performing over 200 semen analysis/year). Of interest, mean bias values for concentration dropped significantly at the third trial for low activity laboratories. In conclusion, lack of agreement of results of semen analysis in Tuscany is mainly because of the activity and the experience of the laboratory. Our study points out the importance of participating in EQC programmes and periodical teaching courses as well as the use of WHO recommended standardized procedures to increase precision and to allow the use of WHO reference values.

Soluble HLA-G molecules in follicular fluid: a tool for oocyte selection in IVF?

Currently, different approaches are used to select oocytes for in vitro fertilization (IVF) procedures, but they do not assure a significant association with the pregnancy outcome. Since several studies have proposed the expression of HLA-G antigens in early embryos to be a possible marker of elevated implantation rate, we have investigated the presence of soluble HLA-G molecules in 50 follicular fluids (FFs). The results have shown soluble HLA-G antigens (sHLA-G) in 19/50 (38%) FFs. Furthermore, we have related the presence of sHLA-G molecules in FFs to detection of the soluble antigens in culture supernatants of the corresponding fertilized oocyte, evidencing a significant relationship (p=1.3 x 10(-6); Fisher exact p-test). Specific ELISA and Western blot approaches identified both HLA-G5 and soluble HLA-G1 molecules in FFs while immunocytochemical analysis indicated polymorphonuclear-like and granulosa cells as responsible for production of sHLA-G1 and HLA-G5 molecules. In contrast, only sHLA-G1 antigens were detected in culture supernatants of fertilized oocytes. Overall, these results suggest a role for sHLA-G molecules in the ovulatory process and propose the FFs analysis for sHLA-G molecule presence as a useful tool for oocyte selection in IVF.

Evaluation of clinical efficacy of three different insemination techniques in couple infertility. A randomized study.

AIM: The aim of the study was to compare the clinical results and efficiency of three insemination technique: intraperitoneal insemination (IPI), fallopian sperm perfusion (FSP) and intrauterine insemination (IUI). METHODS: The experimental design was a prospective, randomized trial. A total of 101 homologous insemination cycles were performed in 71 consecutive couples with unexplained or male subfertility. Couples were randomized to receive IPI or FSP or IUI by predefined tables of randomization and each couple was submitted to the same insemination technique. The primary outcome of the study was the achievement of clinical pregnancy. RESULTS: The results of the study underlined firstly that basal couple composition was not statistically different between the three groups. Moreover, no significant difference in clinical pregnancy rate was observed, despite a clearly positive trend for FSP, especially for unexplained infertility. CONCLUSIONS: Our results showed that the three techniques of insemination IUI, FSP and IPI have similar efficacy on the achievement of clinical pregnancy in couples affected by longstanding infertility.

Embryonic soluble HLA-G as a marker of developmental potential in embryos.

BACKGROUND: In human reproduction, embryo implantation is complex and poorly understood. At present, no single markers are used in routine treatment to assay biochemical functions of the human embryo. Soluble human leukocyte antigen-G (sHLA-G) could be considered a possible marker of embryo developmental potential. It is localized primarily on the extravillous trophoblast, making this antigen a potential mediator of immune interaction at the maternal-fetal interface during gestation. METHODS: Soluble-HLA-G levels were evaluated by an enzyme-linked immunosorbent assay (ELISA) employing monoclonal antibody MEM-G9. It was evaluated in 318 media of single embryo cultures. We correlated the presence of sHLA-G with embryo morphology and the pregnancy obtained in that treatment cycle. RESULTS: No correlation was found between embryo morphology and sHLA-G levels. Pregnancy was observed only when the medium of at least one transferred embryo contained sHLA-G. In 26 out of 66 patients, none of the obtained embryos showed any detectable sHLA-G molecules and no pregnancy occurred. CONCLUSIONS: From our results, we propose sHLA-G as a potential marker of embryo development: the sHLA-G ELISA can be a useful biochemical assay in addition to embryo morphology in embryo selection for transfer in IVF treatment if there are other embryos with the same morphology.

Non-classic sHLA class I in human oocyte culture medium.

Soluble human leukocyte antigen (sHLA) class I molecules have been described in all human fluids. These molecules play a significant role in immune function. sHLA has been shown to produce tolerance and to induce apoptosis in cytotoxic alloreactive T cells. They are also present in the supernatant of many cultured cells. Similarly, non-classic HLA class I antigens in soluble form are present in human fluids. Among these, HLA-G is the most important because of its location in fetal tissue that suggests maternal immunological tolerance of the fetal semiallograft. In our present study we show that using two monoclonal antibodies, w6/32 and TP25.99, in the enzyme-linked immunosorbent assay allows the detection of non-classic sHLA class I molecules in the medium from human embryo cultures. The sample were collected from oocytes cultures. Oocyte donors were 11 women attending the in vitro fertilization program. The results showed a significant association (chi2 = 9.66, p = 0.002) between sHLA antigens and the oocyte cleavage rate measured 48 h after fertilization.

Serum CA-125 values on the day of oocyte retrieval are not predictive of subsequent pregnancy with in-vitro fertilization.

In the clinical management of in-vitro fertilization (IVF) patients it would be very useful to know, before the embryo transfer, whether or not there is a significant chance of pregnancy in that cycle. If low, it would be better to freeze the embryos and postpone the embryo transfer to a subsequent cycle. For this reason, a retrospective study was carried out to investigate the correlations between the serum CA-125 values before embryo transfer and the clinical outcome of that IVF cycle. Women aged <40 years undergoing a complete infertility evaluation including laparoscopy and receiving gonadotrophin-releasing hormone analogue (GnRHa) suppression followed by purified follicle stimulating hormone (FSH) for IVF-embryo transfer were entered into the study. Ninety-seven cycles qualified for evaluation (26 pregnant and 71 non-pregnant cycles). CA-125 concentrations on the day of oocyte retrieval were significantly lower in the pregnant versus non-pregnant cycles in both non-endometriosis and endometriosis patients. To evaluate the existence of a cut-off value of CA-125 which would allow the prediction of a possible pregnancy with sufficient specificity and sensitivity, a receiver operating characteristic curve analysis was performed. This analysis demonstrated the absence of any predictive value of the subsequent pregnancy for CA-125 concentrations. For this reason, and in contrast with previous findings, CA-125 determinations before the embryo transfer in IVF patients do not appear to be a useful tool for clinicians to use in predicting the outcome of IVF in any given cycle.

A sperm survival test and in-vitro fertilization outcome in the presence of male factor infertility.

Several tests based on semen variables have been proposed to predict the fertilization rate in the presence of male factor infertility, but their significance remains unclear. We investigated the utility of a screening test based on sperm survival (SST) to predict the outcome of in-vitro fertilization (IVF) cycles in the presence of male factor infertility. The SST was considered normal when the percentage of motile spermatozoa 24 h after oocyte insemination was > or =50%. The sperm survival test yielded abnormal results in <90% of cycles which were unsuccessful. The sensitivity of the SST was 87% and specificity 65% with a positive predictive value of 90% in the male factor group. We believe that the SST may be a useful predictor of the IVF cycle outcome and we propose its introduction into the routine preliminary evaluation of semen samples in cases of male factor infertility.

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.

In-vivo studies on ovarian insulin-like growth factor I concentrations in human preovulatory follicles and human ovarian circulation.

In some recent hypotheses, the ovary has been indicated as a source of insulin-like growth factor (IGF)-I, with synthesis regulated from local steroidal and non-steroidal substances. We measured IGF-I concentrations in both serum and follicular fluid of women undergoing in-vitro fertilization (IVF) and embryo transfer, in both induced and spontaneous cycles. It was found that serum and follicular IGF-I concentrations were correlated with follicular morphology, oocyte maturity, steroid concentrations and clinical characteristics of IVF cycles. In addition, we measured IGF-I concentrations in both peripheral and ovarian circulation to gain further detailed information on the contribution of the ovary to IGF-I production. The results of our study support the hypothesis that follicular IGF-I is probably derived by diffusion from peripheral circulation and that local production appears unlikely.

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.