1 Department of Obstetrics and Gynecology and 2 Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima and 3 Department of Obstetrics, Hiroshima Prefectural Hospital, Hiroshima, Japan
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi-cho, Minami-ku, Hiroshima 734-8551, Japan. Email: tetsuaki{at}hiroshima-u.ac.jp
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Abstract |
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Key words: acrosome reaction/assisted reproductive technology outcomes/calcium ionophore/complete fertilization failure
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Introduction |
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In this study we investigated whether the calcium ionophore-induced AR is an additional indicator, independent of conventional semen parameters (range, sperm concentration >20 x 106/ml and motility >50% WHO, 1999), for CFF in IVF and pregnancy in IVF and HIT + IUI, in order to determine the best choice of assisted reproduction, using receiver operating characteristic (ROC) curve and multiple logistic regression analyses.
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Materials and methods |
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Semen analysis and ionophore A23187-induced AR
Testing for an ionophore-induced AR was performed as one of the routine investigations during the infertility work-up. A semen sample was obtained by masturbation after 5 days of abstinence. Liquefaction, initiated within 30 min of collection, was achieved by incubation in a 15 ml test tube at 37°C in an atmosphere including 5% CO2. Sperm numbers and motility were determined by a visual estimation using a Makler counting chamber (SEFI Medical, Israel). We measured a combination of capacitation and A23187-induced AR, because sperm with AR after capacitation can fertilize in vivo. Semen was added to modified BiggersWhittenWhittingham medium (mBWW) with HEPES (0.04 mol/l) and 0.5% (w/v) human serum albumin (HSA, fraction V; Sigma Chemical, USA) to give a total volume of 10 ml. After two 5 min centrifugations in this medium at 200 g, the pellet was gently deposited at the bottom of a test tube, and 1 ml of HSAmBWW medium was layered above it. Motile sperm were allowed to swim up into the medium for 1 h. They were then pipetted up from the medium and incubated for an additional 5 h. A 1 mmol/l calcium ionophore A23187 (Sigma Chemical) stock solution in dimethylsulphoxide (DMSO; Sigma Chemical) was prepared and 10 µl aliquots were frozen at 20°C. Before use, this was thawed, dissolved in 0.5 ml of HSAmBWW, and added to 0.5 ml of the motile sperm suspension (2 x 106/ml) to make a final concentration of 10 µmol/l. Incubation was continued for 1 h. The sperm were then centrifuged at 200 g for 5 min. To assess viability, staining also was performed with 1 µg/ml Hoechst 33258 (SigmaAldrich, USA) in 0.5 ml of HSAmBWW for 7 min. The sperm were then centrifuged again at 200 g for 5 min, spread over the entire area of a glass slide, air-dried in a dark room, and fixed in 100% ethanol for 30 min. On removal from the ethanol, the slide was stained with 100 µg/ml of fluorescent isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA; FL1051, Vector Laboratories, USA) for 10 min. The slide was washed in distilled water, dried, treated with an anti-fading agent (15 pg; Bio-Rad, USA), and a coverslip was put on it. The edges were sealed with tape.
Acrosome assessment
We used an epifluorescence microscope (X2F-EFD2; Nikon, Japan) with two filters to observe fluorescence from the Hoechst 33258 (x100, UV filter) and FITCPSA (x100, B2 filter). Sperm were considered non-viable when they showed strong blue-white fluorescence behind the centre of the head with Hoechst 33258; the heads of viable sperm were unstained or stained only lightly. When more than half of the head of a spermatozoon fluoresced brightly and uniformly with FITCPSA, the acrosome was considered intact (Liu and Baker, 1998). Sperm without fluorescence or with a fluorescing band limited to the equatorial segment were considered acrosome-reacted. Of these acrosome-reacted sperm, viable sperm with a fluorescing band limited to the equatorial segment were counted to determine the percentage of AR. The AR was calculated as the number of viable sperm that stained strongly in the equatorial segment divided by the number of viable sperm (Kumagai, 1994
). At least 300 sperm per sample were examined to determine the AR.
One examiner performed the experiment and assessment at the same time and temperature, and under the same atmospheric conditions. Intra-individual variation was confirmed to be acceptable; the mean±SD and coefficient of variation (CV) of the AR between samples of normal fertile volunteers were 31.3±1.9% and 6.1% respectively.
IUI and HIT procedures
Follicular growth was monitored ultrasonographically using a transvaginal probe, and IUI was scheduled to be performed on the day after the largest follicle reached a diameter of 1820 mm in a natural cycle, or 2022 mm in a cycle stimulated using clomiphene citrate. The semen sample for insemination was analysed before and after processing for conventional parameters (volume, sperm count, and motility; WHO, 1999). After the semen had been incubated at 37°C for 20 min, PureCeption (Sage Bio Pharma, USA) was used to separate sperm by centrifugation at 300 g for 30 min (Claassens et al., 1998
). The pellet was washed with 3.0 ml of fresh medium, centrifuged for 5 min at 500 g, and resuspended in a total volume of 0.4 ml for IUI or 50 µl for HIT. Insemination was performed at 3840 h after hCG (Gonatropin®; Teikokuzoki, Japan) administration. The insemination for IUI was carried out using a simple catheter. For insemination for HIT, a sufficiently slender hysterofibrescope (HYF-P; Olympus; outer and channel diameters, 3.6 and 1.2 mm respectively) was inserted through the cervical canal without dilation. Under hysteroscopic observation, the catheter used for insemination was inserted 0.51.0 cm into the oviduct on the side of the dominant follicle. Patients were kept on bed rest for 30 min after IUI and HIT.
Conventional IVF and ICSI procedures
Pituitary secretion was down-regulated by administering 150 µg of buserelin (Suprecur®; Aventis, Japan), a GnRH analogue, as a nasal spray three times daily, beginning in the mid-luteal phase of the cycle preceding the treatment cycle. Pituitary down-regulation was confirmed using both transvaginal scanning and serum estradiol measurement performed on day 7 of the treatment cycle. Injections of hMG (150300 IU/day; Humegon®; Organon, The Netherlands; or hMG Nikken®; Nikken Kagaku, Japan) were then begun. Follicular growth was monitored using transvaginal scanning and serum estradiol concentrations. An i.m. injection of hCG (10 000 IU) was given when at least two follicles were >17 mm in diameter. Oocyte retrieval was performed 36 h after hCG injection. After each oocyte had been cultured for 4 h in human tubal fluid (HTF; Irvine Scientific, USA), it was inseminated with sperm (IVF) or injected (ICSI). For both methods, the sperm used had swum up during 2 h incubation at 37°C with 5% CO2 in the air. ICSI was performed after the cumulus and corona cells had been removed enzymatically with 80 IU/ml hyaluronidase (H-3757, SigmaAldrich). Fertilization was determined by whether two pronuclei could be identified after 20 h. Fertilized oocytes were cultured for 24 h in 1 ml of medium per ovum before transfer into the uterus (maximum, three oocytes) on day 2 after oocyte retrieval. After embryo transfer, vaginal progesterone (300 mg/day) was used to induce the luteal phase. Pregnancy was detected by demonstrating a gestational sac using transvaginal ultrasonography after a positive urine pregnancy test (hCG
50 IU).
Statistical analysis
The results are presented as the mean±SD. P<0.05 was considered significant. The semen parameters, semen count, motility and AR in the CFF and non-CFF groups were compared in the IVF and ICSI groups using the MannWhitney U-test, as were pregnancy and non-pregnancy groups in the IVF, ICSI and HIT + IUI groups.
A receiver operating characteristic (ROC) curve analysis was used to determine the threshold values of the AR for predicting CFF in IVF and pregnancy in IVF and HIT + IUI. Multiple logistic regression analyses were performed using the semen parameters, AR threshold values, reference values of the semen analysis, concentration and motility (WHO manual, 1999; 20x106/ml and 50%) to predict CFF in IVF and pregnancy in IVF and HIT + IUI. The CFF and pregnancy rates in the IVF and ICSI groups were compared using a threshold of 21% using the 2 distribution, as was the fertilization rate in the IVF and ICSI groups using the MannWhitney U-test, with the same threshold of 21%. The pregnancy rates in the IVF and HIT + IUI groups were compared using the threshold value with the
2 distribution.
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Results |
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Semen parameters and assisted reproduction outcomes
Of the 133 cases undergoing IVF and 72 cases undergoing ICSI, 11 (8.3%) and three (4.2%) CFF occurred respectively. The mean semen concentration, motility and AR for CFF and non-CFF in IVF and ICSI are summarized in Table I. Among IVF cases, the AR was significantly lower in the CFF group (P<0.01). The semen concentration was also significantly lower in the CFF group (P<0.05), although the mean values were quite high, compared with the WHO (1999) reference value (20 x 106/ml). Sperm motility in semen was similar in both CFF and non-CFF groups. Among ICSI cases, the AR and sperm motility in semen were similar in both the CFF and non-CFF groups, whereas the semen concentration was significantly lower in the CFF group (P<0.05). The mean value in the CFF group was quite low, compared with the value in non-CFF: 3±3 x 106 versus 42±48 x 106 respectively.
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Discussion |
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The threshold AR value determined using the ROC curve for pregnancy in IVF was 26%. The difference in the threshold values for CFF and pregnancy in IVF, 21 and 26% respectively, suggests that other factors affecting embryo quality for implantation and uterine receptivity have an effect on determining the threshold value for pregnancy. The threshold value of the AR, determined using the ROC curve for pregnancy with in vivo insemination, was 22%. The most important factor for determining the outcome of in vivo insemination might be fertilization. The minimum value of the AR for pregnancy with in vivo insemination was 11.3%. This suggests that those with an AR <10% should not repeat in vivo insemination, but step up to in vitro or split fertilization. In this series, the combined HIT and IUI data were analysed as in vivo insemination data because the AR was not independent of conventional semen parameters in the analysis of each separate treatment procedure. In addition to AR, the total numbers of motile and morphologically normal sperm, numbers of sperm used for insemination, and woman's age significantly affect the pregnancy rate in IUI (Bielsa et al., 1994; Campana et al., 1996
). As for the threshold value for pregnancy with in vivo insemination, more power is needed to verify that the AR is independent of the conventional semen parameters in each modality.
In a previous report, we verified that the percentage of viable sperm with a fluorescing band limited to the equatorial segment was associated with the sperm penetration rate for hamster oocytes and the sperm penetration index more closely than either non-viable sperm with a fluorescing band limited to the equatorial segment or non-fluorescing sperm (Kumagai, 1994). Sperm with a fluorescing band limited to the equatorial segment and sperm with an intact equatorial segment can only bind and fuse with the microvilli of the oocyte membrane (Koehler, 1982
; Talbot, 1982
). It is obvious that the mechanism of the physiological AR induced by the zona pellucida (ZP) differs from the AR induced by other stimuli, including ionophores and progesterone (Liu and Baker, 1994
; ESHRE Andrology Special Interest Group, 1996
). The physiological AR involves activation of several signal transduction pathways, calcium influx, and membrane fusion. Ionophore-induced AR mainly involves a chemical effect on calcium influx (Breitbart and Spungin, 1997
). Although ionophore-induced AR partially reflects a physiological AR process, the AR was the sole predictor of CFF and pregnancy, independent of conventional semen parameters, as part of a routine clinical test.
We did not evaluate sperm morphology as a conventional semen analysis parameter because the validity of assessing sperm morphology, even with the strict criteria used routinely in laboratories, remains controversial (Rhemrev et al., 2001; Mahutte and Arici, 2003
), despite the fact that evaluating sperm morphology using these strict criteria has given initial fundamental information concerning the clinical relevance and predictive value in vivo and in vitro (Grow and Oehninger, 1995
; Ombelet et al., 1995
). A low ionophore-induced AR might be more closely related to the presence of teratozoospermia than to specific defects of the AR (Liu and Baker, 1998
). Conversely, Oehninger et al. (1994)
reported that the non-specific response of abnormal sperm forms, using the strict criteria, to a calcium ionophore was conserved. We did not clarify the relationship between AR and teratosperm in this series, because sperm morphology was not assessed. We plan to use multiple logistic regression analysis to clarify whether the AR is an independent parameter for predicting the outcome of assisted reproduction after considering sperm morphology evaluated using the strict criteria.
In conclusion, ionophore-induced AR provides an additional indicator, which is independent of conventional semen parameters for prediction of CFF in IVF and pregnancy in IVF and HIT + IUI. Assessment of the ionophore-induced AR in patients before commencing assisted reproduction treatments may improve their clinical management.
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Submitted on November 26, 2003; resubmitted on August 16, 2004; accepted on November 2, 2004.
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