Surgical treatment of displaced stress fractures of the femoral neck in military recruits: a report of 42 cases.

BACKGROUND: Stress fracture of the femoral neck (SFFN) is one of the severe complications of military training. Displaced SFFN may result in avascular necrosis of the femoral head (AVNFH). The purpose of this study was to evaluate the results of surgical treatment of the displaced SFFN. METHODS: Forty-two patients with displaced SFFN requiring internal fixation were treated with compression hip screw (17 cases) or multiple cancellous screws (25 cases). Forty-two patients were followed for an average of 5.6 years after operation. At the follow-up evaluation, routine radiographs were used to evaluate the fracture alignment and healing, and bone scan was used for fractures that were suspected of AVNFH. The functional assessments described by Arnold et al. were used to evaluate the functional results of surgical treatment. RESULTS: Seven (28%) of the 25 patients treated with multiple cancellous screws developed AVNFH during the period of follow-up. Of these seven patients, five were treated with prosthetic replacement and two treated with core decompression and bone graft. Three (17.6%) of the 17 patients treated with compression hip screw had AVNFH, and they were all treated with prosthetic replacement. The mean duration between fracture and surgery was significantly longer in patients with AVNFH (5.9 days) than in patients without AVNFH (1.9 days)( P<0.05). Five (71.4%) of the seven patients who presented a varus alignment of the femoral neck developed AVNFH later, and only five (14.3%) of the 35 patients who presented with anatomic or valgus alignment of the femoral neck developed AVNFH during the period of follow-up. Of the 42 patients, 30 (71.4%) had good functional results, four (9.5%) had acceptable and eight (19%) had poor results at the end of evaluation. CONCLUSION: Delayed treatment and postoperative varus alignment were major factors contributing to AVNFH in our series. Early treatment and anatomical fixation of displaced SFFN are essential for diminishing the risk of AVNFH development.

Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing: clinical implications in oocyte freezing--a review article.

Embryo freezing has been a successful practice, but oocyte cryopreservation formerly achieved poorer results. This was mainly due to low rates of survival, fertilization, and development. The major dissimilarities for oocytes to embryos are the character of the plasma membrane, the presence of cortical granules, at the metaphase of meiosis II with the spindle system. In addition, the oocytes must be fertilized by sperm at the appropriate time. To improve the survival rate, a refined slow freezing method with increased sucrose concentration would dehydrate oocytes more sufficiently. Vitrification is another approach to prevent ice crystal formation. Intracytoplasmic sperm injection is used to overcome possible zona hardening from the release of cortical granules. The microtubules of meiotic spindles are vulnerable to the thermal changes and would depolymerize. Cryopreserved oocytes exhibited serious disturbances of the microtubules immediately after thawing. Fertilization of oocytes with disorganized spindles could lead to chromosomal aneuploidy, digyny, and arrest of cleavage. After incubation, the microtubules would repolymerize in a time-dependent way. Normal fertilization and development of cryopreserved oocytes improved after appropriate incubation and timing of insemination, compatible with recovery of the spindles. With the improvement of survival, fertilization, and cleavage, oocyte cryopreservation would gain an imperative role.

Effects of fertilization promoting peptide, adenosine, and pentoxifylline on thawed human sperm.

Fertilization promoting peptide (FPP) and adenosine were demonstrated to be potential modulators of sperm capacitation in mammals. Both FPP and adenosine, by modulating the adenylate cyclase (AC)/cAMP signaling pathway, elicit similar biphasic responses in mammalian sperm (i.e., stimulating capacitation and inhibiting spontaneous acrosome loss). Pentoxifylline, an artificial sperm stimulant, is clinically used to enhance motility of sperm from infertile men. By inhibiting phosphodiesterase, pentoxifylline increases the intracellular cAMP level of sperm, and thus contributes to capacitation, hyperactivation, and acrosome reaction in animal studies. The effects of FPP, adenosine, and pentoxifylline on thawed human sperm are stressed. Chlortetracycline (CTC) fluorescence assessment revealed that none of the 3 reagents improved fertilization ability of post-thawed sperm. Motility studies with computer-aided sperm analyzer (CASA) showed significantly smaller STR (straight-line velocity) and LIN (linearity) in the FPP-treated group at 4 h of incubation p

Distribution of Th1 and Th2 cell populations in human peripheral and decidual T cells from normal and anembryonic pregnancies.

OBJECTIVE: To examine whether maternal immune responses during normal pregnancy are Th2 biased and whether there are specific changes when anembryonic pregnancy occurs. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology at a university hospital. PATIENT(S): We studied 32 pregnant women receiving elective abortions of normal pregnancies and 35 women with anembryonic pregnancies between 6 weeks and 10 weeks of gestational age. INTERVENTION(S): Using the multilabeling capability of three-color flow cytometry, it is possible to measure intracellular cytokines and cell surface markers simultaneously to determine which cells are the cytokine-producing cells. MAIN OUTCOME MEASURE(S): We examined the extent and proportion of mononuclear cells expressing specific T-cell surface markers and cytokines, interferon gamma, and interleukin 4 in the peripheral blood and deciduae. Secreted cytokines in the supernatants after 24-hour culture were also compared. RESULT(S): During the unstimulated status, the proportion of IL-4-secreting cells significantly exceeded that of IFN-gamma-secreting cells in the peripheral blood and decidua in normal pregnancies and was significantly decreased when anembryonic pregnancies occurred. Consequently, the Th1/Th2 ratios were increased during anembryonic pregnancies. However, after 24-hour culture, only another Th2-type cytokine, IL-10, was markedly increased and exceeded IFN-gamma secretion in cultures from both the peripheral blood and decidua in normal pregnancies. CONCLUSION(S): The decidual T lymphocytes are Th2 predominant. When anembryonic pregnancy occurs, this Th2 predominance disappears.

Mouse embryo toxicity of IL-6 in peritoneal fluids from women with or without endometriosis.

BACKGROUND: To determine whether there is a factor (or factors) in the peritoneal fluid of endometriosis patients that impairs embryo growth and embryo implantation. METHODS: Growth and development of two-cell mouse embryos which were cultured in media with peritoneal fluid from women with or without endometriosis and interleukin-1-beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) levels in conditioned media were measured. RESULTS: The blastocyst rate in the non-endometriosis group was 46.4 +/- 31.1%, and that of the endometriosis group was 54.6 +/- 28.7%. Logistic regression analysis using the criteria of blastocyst development in 454 embryos, showed that the peritoneal fluid from endometriosis could promote (p=0.015) but IL-6 could arrest embryo growth to blastocyst (p=0.025). IL-1beta and TNF-alpha levels had no significant effect on blastocyst formation. CONCLUSION: Peritoneal fluid from women with endometriosis was not toxic to mouse embryo development. However, IL-6 in the peritoneal fluid deteriorated the growth and development of mouse embryos.

Increase in the production of interleukin-10 early after implantation is related to the success of pregnancy.

PROBLEM: To study the correlation of interleukin (IL)-10, IL-11 leukemia inhibitory factor (LIF), placental growth factor (PIGF), and transforming growth factor (TGF)-beta and outcome of human pregnancy. METHOD OF STUDY: We prospectively measured the serum levels of these cytokines in patients undergoing in vitro fertilization (IVF) programs. A total of 60 women (non-pregnant, n = 27; early abortions, n = 12; normal pregnancies, n = 21) were enrolled. RESULTS: There was no difference in the cytokines studied on D0 and D14 among the three groups of women. The increase in PIGF from D0 to D14 after human chorionic gonadotropin (hCG) injection was greater in pregnant women than in non-pregnant women; however, the difference did not reach significance (P = 0.068). The increase in IL-10 production from D14 to D21 was significant in women with successful pregnancies compared to women in the abortion group. CONCLUSIONS: This increase in IL-10 may be important in sustaining a normal pregnancy early after implantation.

Increase in the expression of killer cell inhibitory receptors on peritoneal natural killer cells in women with endometriosis.

OBJECTIVE: Malfunction of peritoneal natural killer cells (NK) may result in endometriosis. The present study was designed to determine whether the decrease in NK cytotoxicity occurs at early and advanced stages of endometriosis and is due to the increase in the NK inhibition receptors. DESIGN: A case control study. SETTING: A tertiary-care infertility center . PATIENT(S): A total of 44 women (controls, n = 11; women with early-stage endometriosis, n = 11; and women with advanced-stage endometriosis, n = 22) were included in this study. INTERVENTION(S): Laparoscopic examination. MAIN OUTCOME MEASURE(S): NK cytotoxicity was determined by assay of (51)Cr release against K562 cells, and the expression of killer cell inhibitory receptors (KIR, including NKB1, GL183, and EB6) in NK cells was examined by flow cytometry. RESULT(S): Women with endometriosis showed a decrease in peritoneal NK cytotoxicities against K562 at early and advanced stages of endometriosis. The expression of KIR (NKB1 and EB6) was significantly elevated in the peritoneal NK cells of women with advanced-stage endometriosis compared with controls. KIR (NKB1) was also significantly increased in peritoneal NK cells of women with advanced-stage endometriosis, compared with those of women with early-stage endometriosis. CONCLUSION(S): The results of this study suggest that the decrease in peritoneal NK cytotoxicities against K562 is observed and that this disease may be partially due to the increased expression of KIR on these NK cells.

Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws.

Vitrification of oocytes has been applied recently for humans, but remains elusive. The microtubules of oocytes are vulnerable to cryoprotectants and thermal changes. Using mouse oocytes, the effects of vitrification in open pulled straws (OPS) were investigated on survival, the meiotic spindle, and chromosomes and compared with conventional straws. Mature oocytes were allocated to four groups for exposure to cryoprotectants, vitrification in conventional straws, or vitrification in OPS. They were diluted in stepwise sucrose solutions. Oocytes without treatments were used as controls. The surviving oocytes were stained for meiotic spindles and chromosomes. After dilution, all of the oocytes exposed to cryoprotectants survived. Vitrification sometimes resulted in lysis so that survival using OPS (62%) was significantly (P < 0.05) smaller than that using conventional straws (81%). Oocytes exposed to cryoprotectants or vitrified exhibited serious disturbances of microtubules immediately post-dilution. After 1 h incubation, the microtubules could repolymerize so that the OPS group had significantly (P < 0.05) more normal spindles (78%) than did the conventional straw group (21%). The former also tended to have more compact chromosomes (87%) than did the latter (78%). OPS for vitrification of oocytes achieve more rapid cooling, warming, and dilution and so reduce spindle injury. However, the lower survival rate in OPS needs improvement.

Hormone replacement therapy reverses the decrease in natural killer cytotoxicity but does not reverse the decreases in the T-cell subpopulation or interferon-gamma production in postmenopausal women.

OBJECTIVE: To investigate the immunologic deviations of postmenopausal women before and after hormone replacement therapy (HRT). DESIGN: Prospective study. SETTING: University teaching hospital. PATIENT(S): Seventeen postmenopausal women (study group) and 17 women of reproductive age (control group). INTERVENTION(S): Continuous usage of E(2) valerate 2 mg/d and medroxyprogesterone acetate 5 mg/d in postmenopausal women in the study group. MAIN OUTCOME MEASURE(S): Immunophenotyping with flow cytometry, cytokine production with and without mitogen stimulation of the peripheral mononuclear cells, and a natural killer (NK) cell cytotoxicity test against K562 target cells by the (51)Cr-release assay were performed in the control group and in the study group before, 1 month after, and 6 months after HRT. RESULT(S): NK cytotoxicity, interferon-gamma production, and the T-cell subpopulation were significantly decreased, and the subpopulations of CD3(+)CD25(+) and CD3(+)HLA-DR(+) were increased in the study group before HRT when compared with those in the control group. After HRT was given for 6 months, however, the NK cytotoxicity increased significantly in the postmenopausal women to a value similar to that of the control group. CONCLUSION(S): Women after menopause are prone to impaired immune responses. Nevertheless, some of the impairment can be restored after HRT.

Histopathologic characteristics of a chronic arteriovenous malformation in a swine model: preliminary study.

BACKGROUND AND PURPOSE: The experimental induction of histologic transformations in microvessels of similar caliber to those of nidus vessels of cerebral arteriovenous malformations (AVMs) has not been attempted previously. Our goal was to examine preliminarily the histopathologic characteristics of nidus vessels and the angiographic features of a chronic AVM model in swine. METHODS: AVM models were fashioned from bilateral carotid retia mirabilia of seven swine after the surgical formation of large unilateral carotid-jugular fistulas. One AVM model was made for immediate use, whereas in the other six, follow-up angiography was obtained at varying intervals (2 to 180 days) after model creation. Light and electron microscopy, immunohistochemistry (using monoclonal antibodies against smooth muscle actin and PC10 against proliferating cell nuclear antigen), and histometry were performed on the nidus vessels of three swine: one acutely created, one 2 months old, and one 6 months old. RESULTS: Vascular dilatation and tortuosity of the main arterial feeder and draining vein were evident angiographically as early as 4 days after AVM creation, and were maximal in the 6-month-old model. Compared with the acutely created nidus vessels, those in the two chronic models revealed disrupted and attenuated elastica and intimal hyperplasia that was focal ("cushions") or generalized, leading to luminal occlusion. Variable numbers of cells in the tunica media of chronic nidus vessels contained smooth muscle actin. PC10/proliferating cell nuclear antigen immunoreactivity was observed in the endothelium and subendothelial layers. Histometry showed increases in intimal hyperplasia and medial thickness in the chronic vessels. CONCLUSION: Nidus vessels in this chronic swine AVM model exhibited striking histologic changes similar to those seen in cerebral AVMs. The induced vessel growth seen angiographically and histologically in components of the chronic AVMs was consistent with the presence of persistently raised intravascular hemodynamic loads. This preliminary feasibility study suggests that the realistic histologic characteristics of this chronic AVM model are an attractive feature, and if confirmed in future, more comprehensive, studies would be of benefit in accurate histopathologic interpretation of the effects of superimposed experimental embolotherapy or radiosurgery. This model may provide a useful experimental tool to study the dynamic cellular and tissue events that dictate the development and natural history of AVMs.

Luteinizing hormone up-regulates the expression of interleukin-1 beta mRNA in human granulosa-luteal cells.

PROBLEM: Previously, we observed that follicular fluid obtained from patients with premature luteinization contained elevated interleukin-1 beta (IL-1 beta) levels. In this study. we aimed to examine the effects of luteinizing hormone (LH) on IL-1 beta expression and IL-1 beta on steroidogenesis in human granulosa-luteal cells. METHOD OF STUDY: Human granulosa-luteal cells were obtained during oocyte retrieval. The cells were treated with either LH or IL-1 beta and subsequently were examined for the level of IL-1 beta transcript. The conditioned media were examined for IL-1 beta protein and steroid hormone levels. RESULTS: LH (250-500 mIU/mL) up-regulated the expression of IL-1 beta mRNA (up to a 4-fold increase over control; P<0.05) in the granulosa-luteal cells. IL-1 beta (5-50 ng/mL) increased the basal, but not LH-dependent, progesterone production from these cells in a dose-dependent manner after 96 and 144 hr of culture (P<0.05). However, an inhibitory effect of IL-1 beta on LH-dependent estradiol production was observed (up to 20% decrease, P<0.05). CONCLUSIONS: LH is capable of stimulating IL-1 beta transcript expression in human granulosa-luteal cells and may regulate ovarian steroidogenesis, at least partly through the activation of IL-1 beta.

The expression of killer cell inhibitory receptors on natural killer cells and activation status of CD4+ and CD8+ T cells in the decidua of normal and abnormal early pregnancies.

The establishment of the human placenta in early pregnancy is characterized by the presence of large numbers of natural killer cells within the maternal decidua. These NK cells have an unusual phenotype, CD3- CD16- CD56(bright), distinguishing them from peripheral blood NK cells. They may control trophoblast migration and placentation. Using a panel of monoclonal antibodies to several members of the KIR family and flow cytometry, we found that KIRs are expressed on decidual NK cells. There is variation in both the percentage of cells expressing a particular receptor and the density of receptor expression between decidual NK cells from different individuals. In anembryonic pregnancy, the proportions of decidual NK cells with a particular KIRs (GL183 and EB6) decreased significantly when compared with normal pregnancy (p = 0.01 and 0.01, respectively), raising the possibility that these NK receptors may be involved in recognition of the allogeneic fetus by the mother at the implantation site. In the decidua, more CD4+ and CD8+ T cells expressed CD69 and HLA-DR than in blood, indicating that T cells are regionally activated during early pregnancy. When compared with normal pregnancy, decidual HLA-DR+CD4+CD3+, CD69+CD8+CD3+ and HLA-DR+CD8+CD3+ T lymphocytes are significantly increased in anembryonic pregnancy. The over-activation of decidual T cells during anembryonic pregnancy may thus contribute to the increased NK cytotoxicity activity.

T lymphocytes and cytokine production in ascitic fluid of ovarian malignancies.

The activation status of T lymphocytes and the presence of various cytokines in ascitic fluid were examined to test peritoneal immunity in women with ovarian malignancies. Peripheral blood and peritoneal fluid were collected from 12 patients with primary ovarian cancer with ascites and 27 normal control subjects during laparoscopic examination. Lymphocyte subpopulations and the expression of activation markers on T lymphocytes were analyzed by dual-color flow cytometry. The concentrations of various cytokines and soluble interleukin (IL)-2 receptor-alpha were measured. CD8 T lymphocytes were the main component of peritoneal lymphocytes. CD69 and HLA-DR, but not CD25, were highly expressed on peritoneal T lymphocytes compared to those in peripheral blood. In ascitic fluid of ovarian malignancies, CD4 T lymphocyte concentrations were further decreased, resulting in a decreased CD4/CD8 ratio. Decreased expression of CD69 and CD25 was also noted on T lymphocytes from ascites compared with T lymphocytes in normal peritoneal fluid. IL-1b, tumor necrosis factor-alpha, IL-6, and soluble IL-2 receptor-alpha concentrations were increased significantly in the ascitic fluid of women with ovarian cancer. The decrease in activation markers on T lymphocytes is suggestive of an immunosuppressive state, despite the presence of abundant stimulatory cytokines. The immunosuppression may be multifactorial, attributed, in part, to the increased concentrations of soluble IL-2 receptor-alpha and other inhibitors.

Increase in the production of interleukin-6, interleukin-10, and interleukin-12 by lipopolysaccharide-stimulated peritoneal macrophages from women with endometriosis.

PROBLEM: To verify whether the peritoneal macrophage (PM) is activated in endometriosis. METHOD OF STUDY: We examined the synthesis of nitric oxide (NO), total antioxidant, interleukin (IL)-6, IL-10, and IL-12 by cultured PMs, which were either unstimulated or stimulated with lipopolysaccharide (LPS), from women with endometriosis (early, n = 12; advanced, n = 11) or without endometriosis (n = 13). RESULTS: After stimulation with 2 ng/mL LPS for 24 hr, PMs from women with early-stage endometriosis secreted more NO, IL-6, and IL-10 than the controls. Higher IL-12 levels were noted in women with advanced endometriosis when compared with the controls. After 2 ng/mL-LPS stimulation for 24 hr, we also detected higher total antioxidant levels in the advanced-endometriosis group than those in the early-endometriosis group. CONCLUSION: The increased production of IL-6, IL-10, and IL-12 by stimulated PMs confirmed previous observations that the PM is the principle source of these cytokines in peritoneal fluid.

Surgery for symptomatic Freiberg's disease: extraarticular dorsal closing-wedge osteotomy in 13 patients followed for 2-4 years.

From 1992 through 1995, we have treated 13 patients (10 men) with Freiberg's disease by debridement and dorsal closing-wedge osteotomy of the metatarsal neck. The lesion was located in the second metatarsal head in 10 patients and in the third metatarsal head in 3. After osteotomy, the lesion was away from the joint, so that the smooth and healthy articular cartilage of the metatarsal head faced the phalangeal cartilage. The average follow-up period was 40 (28-54) months. The subjective outcome was good or excellent in 11 patients, fair in 1, and poor in 1. We found MRI useful in determining the extent of the lesion when planning correction.

Controlled ovarian hyperstimulation and intrauterine insemination in subfertility. How many treatment cycles are sufficient?

OBJECTIVE: To examine the combined effects of factors influencing the pregnancy rates following controlled ovarian hyperstimulation (COH) and intrauterine insemination (IUI). STUDY DESIGN: After utilizing the couple as the unit of analysis, 416 couples undergoing COH and IUI with the husband's sperm were studied retrospectively. RESULTS: The woman's age, postwash total motile sperm counts and number of IUI treatment cycles were three major factors that affected the results of IUI. The predicted cumulative pregnancy rates under different values of the above three factors were therefore calculated using multiple logistic regression analysis and were compared to the pregnancy rate achieved by a cycle of in vitro fertilization and tubal embryo transfer. CONCLUSION: Three treatment cycles of IUI are optimal for women aged < or = 34 with postwash total motile sperm counts > 5 x 10(6). For women either aged > or = 35 or with postwash total motile sperm counts < 5 x 10(6), the cumulative pregnancy rates achieved by IUI are extremely low, and other means of assisted reproduction are recommended for couples in those categories. However, this conclusion pertains to our clinics, and other institutions should establish their own data.

Effects of repeated abdominal paracentesis on uterine and intraovarian haemodynamics and pregnancy outcome in severe ovarian hyperstimulation syndrome.

The aims of this study were to investigate the effects of paracentesis on uterine and intraovarian haemodynamics by colour Doppler ultrasound and the influences of repeated paracentesis on pregnancy outcome in severe ovarian hyperstimulation syndrome (OHSS). Forty-one abdominal paracenteses were performed on seven pregnant women with tense ascites and eight thoracocenteses were performed on three pregnant women with pleural effusion. Pulsatility index (PI) and maximum peak systolic velocity (MPSV) of uterine and intraovarian arteries were measured before and after each intervention. The mean PI of uterine arteries was decreased significantly after paracentesis, but not after thoracocentesis. Furthermore, uterine PI was decreased in 13 out of 14 (92.9%) paracenteses with <2500 ml ascites removed, compared with eight out of 13 (61.5%) with >2500 ml ascites removed. After paracentesis, there were no significant changes in the intraovarian PI and MPSV in either group. The 24-hour urine output increased significantly in the paracentesis group, but not in the thoracocentesis group. There were no significant changes in haematocrit and electrolytes as a result of paracentesis. However, gradual falls in serum total proteins and albumin concentrations were observed in all patients after repeated paracentesis, necessitating post-paracentesis albumin infusion. There was no significant difference in miscarriage rates between the two groups. We conclude that repeated abdominal paracentesis increases uterine perfusion and has no adverse effects on pregnancy outcome in severe OHSS. Extraction of 2500 ml of ascitic fluid did not impair uterine perfusion.

Cornual pregnancy after IVF-ET. A report of three cases.

BACKGROUND: Ectopic pregnancy is a well-known complication of in vitro fertilization/embryo transfer (IVF-ET). Although ectopic pregnancy after IVF-ET in patients with prior bilateral salpingectomy is uncommon, it can occur and may be a cornual implantation. Because of the possibility of early rupture with severe hemorrhage, special attention to cornual pregnancy after IVF-ET is warranted. CASES: Three cases of cornual pregnancy occurred after IVF-ET. Two of these patients had prior bilateral salpingectomy, whereas another had prior tuboplasty for tubal disease. CONCLUSION: Patients who had prior salpingectomy or tuboplasty with cornual patency undergoing IVF-ET are at risk for cornual pregnancy. Close monitoring of pregnancies in these patients is important to prevent a deleterious delay in treatment of a cornual pregnancy.

Spermatozoa recovered by IxaPrep gradient have improved longevity and better motion characteristics than those by Percoll gradient.

To compare the effectiveness of various sperm preparation methods, we examined sperm concentration, percentage of progressive motility, recovery rate, and various motion characteristics in 32 semen samples after Percoll and IxaPrep preparations. Except for sperm concentration, which was much higher after IxaPrep preparation, no statistical differences existed between these two methods in terms of motile sperm concentration (MSC), recovery rate, mean curvilinear velocity (VCL), mean straight-line velocity (VSL) and mean amplitude of lateral head displacement (ALH). Among 22 samples that were allocated in a 37 degrees C, 5% CO2 incubator, the mean MSC after 3 hours was significantly higher following IxaPrep preparation than following Percoll preparation (63.2 x 10(6)/mL vs. 42.8 x 10(6)/mL, p < .03). VCL and VSL were much higher after IxaPrep preparation than after Percoll preparation, both at 3 hours (p < .01) and at 24 hours (p < .03). ALH was also higher after IxaPrep preparation than after Percoll preparation, and it was statistically significant after 3 hours of incubation (p < .03). We conclude that the IxaPrep gradient may be adopted as the preferred method to the Percoll gradient in preparing spermatozoa for assisted reproduction because of the spermatozoa's persistent and better motion activities after incubation.