Asian Pacific Journal of Reproduction

Pages 85-90

Authors: Jeriz Anne S. de Guzman, Reymond John L. Beltran, Raquel Rubio, Gliceria B. Ramos

Objective To examine the potential of pre-gestational intake of Laportea interrupta L. (L. interrupta) leaf decoction as an aid for fetal-maternal health by determining its influence on embryonic implantation and growth, placental labyrinth vasculo-angiogenesis, and junctional zone morphology. Methods Eight-week-old female mice were divided into groups and fed daily with 3.5 g food/mouse. The control was given drinking water, the treatment groups, low (LC), medium (MC), and high (HC) concentrations, were given 25%, 50%, and 100% v/v stock solution, respectively for 14 days, prior to mating. Pregnant mice were sacrificed at 14.5 days post-coitus. The uteri and placentae were collected and weighed; implantation sites were counted as either viable or resorbing. The estimated weight (g)/ embryo, as a function of the number of implantation sites, was evaluated. The histology of placental labyrinth angiogenesis and junctional zone morphology was examined. Results The viable site ratios increased as leaf decoction concentration increased. This was most significant in the HC group (P<0.05). The HC group exhibited increase in the estimated embryo weight and implantation sites, and placental labyrinth with very prominent blood vessels. There was lesser depletion of junctional zone in all treatment groups with large blood vessels and glycogen cells that were more apparent in MC and HC group than those of the control and LC groups. Conclusions Pre-gestational consumption of L. interrupta leaf decoction in high concentrations demonstrated its potential to support fetal-maternal health.

Pages 96-99

Authors: Sutrisno Sutrisno, RR Catur Leny Wulandari, Dwi Wahyu Wulan Sulistyowati, Ratna Feti Wulandari, Endang Sri Wahyuni, Yuyun Yueniwati, Sanarto Santoso

Objective To investigate the effect of genistein on proinflammatory cytokines, NF-κ B activation, and estrogen receptor-β expression in a mice model of endometriosis. Methods Forty female mice (Mus musculus) were divided into eight groups (n=5 each), including the control (untreated) group, endometriosis group, and the endometriosis groups were given various doses of genistein (at doses of 50; 100; 200; 300; 400; 500 mg/day). Analysis of TNF-α, IL-1β, IL-6, and IL-8 level were done by ELISA technically. Analysis of estrogen receptor-β and NF-κB were done by immunohistochemistry. Results The level of TNF-α, IL-1β, IL-6, and IL-8 were significantly higher in the EM group compared to the untreated control group (P<0.05). All doses genistein significantly prevented EM-induced increase in TNF-α level (P<0.05), but only at dose of 300 mg/day reach the level in the control group (P>0.05). These increased levels of IL-1β, IL-6, adn IL-8 in the EM group were significantly reduced by all doses of genistein. There were significantly (P<0.05) increased estrogen receptor-β expression and NF-κB activation in EM group compared to untreated group. Only first and fourth dose significantly (P<0.05) decreased the estrogen receptor-β expression compared to the EM group, to reach a level in the control group (P>0.05). All doses genistein significantly prevented EM-induced increase in NF-κB activation (P<0.05), to reach the expression on control group. Conclusions In conclusion, genistein prohibits the increase in proinflammatory cytokines, NF-κB, and estrogen receptor-β expression in a mice model of endometriosis.

Pages 106-111

Authors: Arturo Morales Martinez, Luis H. Sordia-Hernández, Juan A. Morales, Martha Merino, Oscar Vidal, Manuel R. García Garza, Otto Valdés

Objective To determine whether subjects suffering from oligoasthenozoospermia would benefit from antioxidant treatment with resveratrol, a natural-occurring polyphenol, and hydrogen sulfide. Methods A randomized controlled clinical trial involving 54 men with Oligoasthenozoospermia. We randomly assigned resveratrol (n=18), SG 1002 (n=18), and placebo (n=18) for 75 days. Sperm analysis was performed after treatment. Statistical analysis was made with chi square test. Results When compared to the placebo treated group, SG1002 treatment led to an increase in sperm concentration (11.18 × 106 vs. 17.01 × 106, P < 0.05), sperm motility (10.06 × 106 vs. 20.06 × 106, P<0.05) and motile forms recovery (0.33 × 106 vs. 1.62 × 106, P<0.05). Resveratrol treatment did not significantly affect any of the parameters. Conclusions SG1002 may reverse oligoasthenozoospermia. It seems to be more potent antioxidant than resveratrol. This findings need be supported by further clinical investigation.

Pages 116-122

Authors: Reyhane Hoshyar, Zabihollah Mohaghegh, Nihad Torabi, Aliyeh Abolghasemi

Objective To investigate various therapeutic effects of medicinal plant Ziziphus jujube (Z. jujube), such as antibacterial, antiviral, anticancer, anti-inflammatory and antioxidant properties. Methods The present study was conducted to assess the beneficial effects of aqueous extract of Z. jujube fruit on the proliferation of breast cancer cells by MTT assay and the antioxidant by FRAP, haematological and biochemical alterations caused by NMU-induced mammary carcinogenesis in rats. The rats were divided into five groups, control group A (n=10), Z. jujube control group B (n=10), NMU control group C (n=15), Z. jujube treatment group D (n=15) and Z. jujube prevention group E (n=15). At the end of the experimental period, all the animals were euthanized and blood was collected by heart puncture. Results The Z. jujube revealed a dose- and time-dependent cytotoxic effect against an MDA-MB-468 cell line and its treatment significantly (P<0.05) increased the total antioxidant capacity when compared to the NMU control group C. Z. jujube exhibited a preventive effect against anaemia, lymphocytosis and neutrophilia in group D and group E, when compared to group C. Biochemical analyses showed normal levels of enzymes of the liver in the Z. jujube treated groups: B, D and E rats, whereas NMU control group C showed significant (P<0.05) decreases in ALT, AST, albumin and total protein levels, and significant (P<0.05) increases in ALP and LDH. Conclusions These findings indicate that the Z. jujube ameliorates the adverse effects of NMU carcinogenesis and could be useful for treating mammary tumours in humans.

Pages 129-134

Authors: Carlos Granados-Echegoyen, Rafael Pérez-Pacheco, Alfonso Alexander-Aguilera, Luicita Lagunez-Rivera, Nancy Alonso-Hernández, Eleazar de Jesús Chairez-Martinez

Objective To perform the toxicological evaluation of aqueous and ethanol extract of dried leaves of Pseudocalymma alliaceum (P. alliaceum) in male Wistar rats by oral administration for 14 days, and to determine the biochemical and haematological status of blood. Methods The animals were completely randomized into four groups of three rats each. Results No deaths were reported after oral administration of the extracts, no physical signs of toxicity or adverse effects were observed. Hematological indices of red blood cell count, hemoglobin concentration and mean corpuscular hemoglobin showed no significant abnormality; however, white series levels decrease presenting a leukopenia. Glucose, creatinine and albumin increased, while urea decreased; aspartate aminotransferase values decreased with the aqueous extract at 50 and 100 mg/kg and increased with dose of 200 mg/kg, in contrast ethanol extract caused an increase in this parameter to the doses used. The alanine aminotransferase decreased with aqueous extract and increased with ethanol extract. Triglycerides decreased when used aqueous extract and reduced with ethanol extract at 100 and 200 mg/kg, in contrast to 50 mg/kg decreased to be compared with control group. Conclusions The daily intake of P. alliaceum did not produce acute toxicity to 50 mg/kg which may be interpreted as toxic signs or biological damage, but liver and renal function changes at dosages of 100 and 200 mg/kg; however, the reduction ability of white blood cells count could be used as a basis for specific studies on the treatment of patients with leukemia.

Pages 140-146

Authors: Yanhe Liu, Christopher Copeland, Vincent Chapple, Peter Roberts, Katie Feenan, Phillip Matson

Objective To investigate the relationship between elevated serum progesterone levels (EP) on the day of ovulatory trigger, live birth rates, and the growth of resulting embryos. Methods A total of 836 in vitro fertilization (IVF) cycles with 4 478 embryos in conventional culture were retrospectively analyzed, together with an additional 90 IVF cycles producing 618 embryos from culture and assessment using the Embryoscope™ time-lapse system. Results In cycles using conventional culture, serum progesterone per follicle ≥14 mm (median 0.42 nmol/L/follicle, range 0.05-3.50 nmol/L/follicle) was a significant negative predictor of live-birth (ROC AUC = 0.395, 95% CI 0.345-0.445; P=0.000) as were progesterone/estradiol ratio (0.442, 0.391-0.494; P=0.027) and progesterone per oocyte (0.374, 0.326-0.421; P=0.000) but not progesterone alone (0.470, 0.419-0.521; P>0.05). Women with an EP/follicle (>0.42 nmol/L/follicle) had reduced live birth rates if they were ≥35 yrs (14.4% vs. 24.2%, P<0.05) but not <35 years (35.3% vs. 37.4%, ns). Despite reduced pregnancy rates, cycles with EP/follicle in women ≥35 years produced similar proportions of “good” and “top” quality embryos in conventional culture compared to women with low progesterone/follicle, and no difference in abnormalities of cleavage (direct cleavage or reverse cleavage), multinucleation or timings of division (pronuclear fading to 2-cell, 3-cell, 4-cell and 5-cell; cc2 and S2) observed with time-lapse videography. Conclusions EP/follicle ≥14 mm (>0.42 nmol/L/follicle) adversely affects embryo implantation in women aged ≥35 years, but not <35 yrs. However, no adverse features were seen in the embryos from these affected cycles in terms of morphological appearance, abnormal patterns of cleavage, or morphokinetic timings.

Pages 153-156

Authors: CT Sulaiman, A Arun, EM Anandan, CR Sandhya, Indira Balachandran

Objective To develop analytical methods for the isolation and structural identification of poly phenols including phytoestrogens in Mensokot tablet, a herbal proprietary medicine. Methods Isolation consisted of an ultrasound-assisted extraction, followed by acid hydrolysis and a final liquid-liquid extraction step in diethyl ether. Identification and structural characterisation was done by liquid chromatography coupled with Q-TOF-ESI-MS/MS analysis. Results Phytoestrogens such as Coumestrol, Genistein and Glycitein have been identified in Mensokot tablet along with several other flavonoids. Conclusions In the present research, a rapid HPLC-MS/MS method has been developed for the identification of phytoestrogens and other flavonoids from an Ayurvedic proprietary medicine. Phytoestrogens are considered to play an important role in the prevention of cancers, heart disease, menopausal symptoms and osteoporosis.

Pages 166-168

Authors: M Aziken, J Osaikhuwuomwan, A Osemwenkha, O Iribhogbe, A Orhue

Gonadal dysgenesis represents a congenital developmental disorder of the reproductive system, with its main gynaecologic manifestations being amenorrhea and infertility. We present a unique case of pure gonadal dysgenesis in an ‘about to be’ married lady resident in a society where high premium and success in marriage is dependent on childbirth. With astute evaluation and counseling, assisted reproductive technology (ART) was safely and successfully used in this case with eventual triplet pregnancy and delivery. Our index experience shows that situations with compromised fertility the availability and access to ART aids effective treatment planning and births a re-invigorated hope for family life.

Pages 1-12

Authors: P Perumal, JK Chamuah, AK Nahak, C Rajkhowa

Objective To assess the effect of melatonin (MT) at different seasons of the year on sperm motility, viability, total sperm abnormality, acrosomal membrane, plasma membrane and nuclear integrity, antioxidant profiles such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and total antioxidant capacity (TAC), enzymatic profiles such as aspartate amino transaminase (AST), alanine amino transaminase (ALT) and biochemical profiles such as malondialdehyde (MDA) production. Methods Total numbers of 80 ejaculates (20 ejaculates in each season) were collected twice a week from mithun bulls and semen was split into five equal aliquots, diluted with the tris egg yolk citrate (TEYC) extender. Group 1: semen without additives (control), group 2 to group 5: semen was diluted with 1 mM, 2 mM, 3 mM, and 4 mM of melatonin, respectively. These seminal parameters, antioxidant, enzymatic and biochemical profiles were assessed at liquid storage of mithun semen (5 °C). Simultaneously, retrospective study was conducted on birth rate of calf at different seasons from 2002 to 2012 from farm birth register of mithun farm. Results Inclusion of melatonin into diluent resulted in significant (P<0.05) decrease in percentages of dead spermatozoa, abnormal spermatozoa and acrosomal abnormalities at different seasons of the year as compared with control group. Additionally, spring season has highest seminal and antioxidant profiles followed by autumn and winter season, whereas lower values were observed in ejaculates collected from summer season. Similarly retrospective study revealed that highest birth rate was in winter followed by autumn, spring and summer season and breeding was occurred in spring, winter, summer and autumn season, respectively with gestation period of 270–290 days. Conclusions The result of present study indicates that the melatonin protects seminal and antioxidant profiles varied in different seasons, semen quality also varied from different seasons and was highest in spring and lowest in summer season. It was concluded from the present study that breeding takes place throughout year but the peak breeding season is from November to April (spring and winter season) in mithun.

Pages 17-21

Authors: Andrey Lyashenko

Objective To improve the indicators of motility, survival and fertilizing ability of spermatozoa by optimizing temperature factors and the duration of exposure at unfreezing straws. Methods Straws by volume 0.25 mL were thawed at water bath temperatures at 65 °C, 67 °C and 70 °C for 6–7 seconds and at 75 °C for 4–6 seconds. Impact of exposure time and temperature thawing in the water bath on motility and survival of spermatozoa were studied. Results Studies indicate that for the procedure of defrost water bath straws in seven seconds for temperature conditions of 65 °C, 67 °C and 70 °C, indicators of progressive motility and absolute survival rate were significantly higher than for the control group an average on 11.4 % (P<0.01). Optimum exposure time (6–7 seconds) and temperature range (65–70 °C) defrosting semen doses were defined. Conclusions Owing obtained the positive result, method of thawing was developed which increases the indicators of motility, survival and fertilizing capacity of bull semen.

Pages 26-31

Authors: Reda I. El-Sheshtawy, Gamal A. Sisy, Walid S. El-Nattat

Objective To examine the effect of different concentrations of trehalose or sucrose (50 or 100 or 200 mM) on post-thawed quality of bull semen, cryo-preserved in Tris-citric acid-egg yolk-fructose (TCYF). Methods Semen samples were diluted in TCYF extender, TCYF + trehalose (50, 100 and 150 mM/L) or TCYF + sucrose (50, 100 and 150 mM/L) to ensure 60 million motile spermatozoa mL-1, cooled slowly up to 5 °C and equilibrated for 4 h. Semen was packed into 0.25 mL polyvinyl French straws. The straws were placed horizontally on a rack and frozen in a vapor 4 cm above liquid nitrogen (LN2) for 10 minutes then dipped in liquid LN2. Frozen straws were thawed at 37 °C for 1 min. The parameters studied were sperm motility, sperm viability, sperm abnormality, sperm membrane integrity (HOST), percent of normal intact acrosome and DNA fragmentation. Results The output data demonstrated that addition of 50–100 mM of trehalose or sucrose/L TCYF after chilling at 5 °C had significantly (P<0.0001) ameliorated motility, membrane integrity, viability, abnormal morphology, and acrosome integrity % compared to control diluted semen while 50 mM of trehalose/L, and 50–100 mM of sucrose/L to TCYF diluent had significantly (P<0.0001) improved after thawing motility (43.00,% 45.00% and 41.00%, respectively), membrane integrity (67.40%, 67.80% and 69.40%, respectively), life sperm % (70.20%, 69.40% and 71.40% respectively), and acrosome integrity percentages (56.40%, 58.80% and 55.80% respectively) compared to the control tris-base diluent, while diminishing the abnormal sperm morphology (6.20, 3.80 and 3.80 respectively) and DNA fragmentation (3.60%, 3.80% and 3.80% respectively). Besides, the addition of 100 mM of trehalose/L to tris-base diluent has also a promising effect when added to the tris-base diluent concerning the above parameters. Conclusion It is finally concluded that the addition of 50–100 mM trehalose or sucrose/L to TCYF have a beneficial effect in chilling diluted bull semen, while the use of 50 mM trehalose or 50–100 mM sucrose had their benefits on freezing-thawing of extended semen.

Pages 37-40

Authors: Augustave Kenfack, Ferdinand Ngoula, Paul Désiré Womeni Dzeufiet, Omer Bébé Ngouateu, Tsambou Megnimeza Astride Martine, Judith Kegne Chombong, Guylène Marie Zambou Zeukeng, Isabelle Leinyuy Nyuysemo, Arthenice Jemima Noumamo Guiekep, Tah Patience Nain, Pierre Kamtchouing, Joseph Tchoumbou, Narcisse Bertin Vemo

Objective To study the effects of chlorpiryphos-ethyl (CE) on male fertility. Methods Rats were gavaged daily from 30 to 120 days old with four doses of CE (10.50; 5.25; 3.50 and 0.00 mg/kg) and thereafter followed up for 90 additional days. Following this period of observation, each male rat was submitted to a fertility test with two virgin females. Male were then sacrificed and organs collected while females were followed up till delivery. Results The weights of the testis and epididymis were lower (P<0.05) in rats treated with 10.50 mg/kg CE dose compared to controls. A decrease in the number of sperm per cauda epididymis was recorded in rat given the highest dose of CE with respect to the control value. The sperm motility was lower (P<0.05) in all CE-treated groups compared to the controls. Severe histological abnormalities were observed in testes of treated rats. The males exposed to the highest dose of CE did not produce any offspring. Conclusion The effects of CE on reproductive system were persistent 90 days after the end of exposure.

Pages 44-48

Authors: Ibrahim A. Abdelazim, Walid Farok Elsawah

Objective To detect the prevalence of metabolic syndrome (MS) among infertile women with polycystic ovary syndrome (PCOS). Methods Two hundred and twenty infertile PCOS women were included in this prospective cross section study. Diagnosis of PCOS was based on at least two of ESHRE/ASRM criteria and diagnosis of MS was based on at least three of NCEP ATP III criteria. A standard questionnaire was used to document length of menstrual cycles, personal and family history of medical disorders. Signs of androgen excess and insulin resistance were noted in the physical examination. Anthropometric measurements were done to measure waist circumference, hip circumference and body mass index (BMI) was calculated. Overnight fasting blood sample and a 75 gram oral glucose tolerance test, TSH, prolactin, total testosterone, SHBG and lipid profile levels were evaluated in all studied PCOS women. Trans-vaginal ultrasound was also done to measure; ovarian volume and number of follicles in both ovaries. Results The prevalence of MS in studied PCOS women was 30.5% (67/220). There is strong positive correlation between prevalence of MS and both age and BMI of the studied PCOS women. Logistic regression analysis showed that; the age > 25 and waist-hip ratio ⩾ 0.85 were powerful predictors for the prevalence of MS in PCOS women. Conclusion The prevalence of MS was 30.5% in the studied PCOS women. The age above 25 years and waist-hip ratio ⩾ 0.85 were powerful predictors for prevalence of MS in PCOS women.

Pages 54-60

Authors: Abdelhanine Ayad, Mourad Salaheddine, Kamal Touati, Mokrane Iguer-Ouada, Hama Benbarek

Objective To evaluate the effectiveness of norgestomet Crestar® for synchronization of oestrus and its effect on pregnancy rate in cows. Methods after a waiting period (∼90 days postpartum), Holstein-Frisian females kept in different dairy farms were inseminated for the detection of estrous behavour (n =133). Females were allocated randomly to treated (n =69) or control (n =64) groups. The treated animals received the Crestar® treatment during 9–10 days. After implant removal, the treated animals were artificially inseminated (AI) for the detection of standing estrous behaviour. Blood samples were collected on Days 0, 21 and 30 after service. Plasma progesterone (P4) and pregnancy-associated glycoprotein (PAG) levels were determinate by radioimmunoassay. Results the study showed a higher rate of heat synchronization in the treated group (94%) compared to the control group (86%). Out of 133 cows inseminated, thirteen (4 treated and 9 control) had a plasma progesterone concentrations higher than 1.0 ng/mL at service. The plasma PAG levels at Day 30 after AI were significantly lower in the treated group (7.13 ± 2.29) compared to the control group (8.37 ± 2.30). Pregnancy rate was 28% (16/57) and 42% (23/54) for treated and untreated group, respectively. There were no significant differences in the pregnancy rates of different parity in both groups. Conclusion The norgestomet Crestar® treatment carried out during 9–10 days did not improve reproductive performance in dairy cattle. However, these data demonstrate that this hormonal protocol can be used to obtain high oestrus cow rates to initiate correct timing of insemination.

Pages 65-69

Authors: R Asadpour, MH Asadi, R Jafari-Joozani, GH Hamidian

Objective To use PCR to investigate the presence of fetal SRY gene in the ovine cervical secretions and maternal blood plasma, and to assess predict fetal sex at different times of gestation in the ewe. Methods Fetal DNA was isolated from blood plasma and cervical secretions of 32 pregnant ewes during the 6 to 21 week of gestation. Overall, 15 male and 17 female fetuses were included in this study. After DNA extraction, the PCR amplified a 280 bp fragment from the X-chromosome and a 217 bp fragment from the Y-chromosome based on a sex-related polymorphism in the amelogenin locus. Results The presence of fetal Y-chromosome was confirmed in 11 out of 15 cervical mucus and 12 out of 15 blood plasma samples collected from sheep with male fetuses. The sensitivity and specificity of tests were 70% with false negative results. Conclusion This is the first report on validating the presence of fetal DNA material in the ovine cervical mucus and its potential usefulness for fetal sexing.

Pages 74-77

Authors: JE Rodríguez, A López, Instituto Sexológico Murciano

Objective To determine if they can produce increases in the IELT using a masturbator in subjects with premature ejaculation, producing improvements in the quality and satisfaction of the sexuality of the patient. Methods To carry out the study it will arrange of the utilization of a masturbation device aid for six weeks that they will use at least during 5 minutes without overcoming the half daily hour in the manual masturbation. The measures of results will be the differentiation of the basal and final IELT by means of the average of both results, obtaining the differential percentage of the above mentioned measures. Results There was obtained a sample of 9 heterosexual participants of ages included between 20 and 42 years. The time of latency increased from the first week of treatment producing up to 57 % of increase in the IELT in the masturbation and up to 79.9 % of improvement during the coitus. Conclusions The use of a masturbation aid (FLIP HOLE) has shown to be a useful tool in increase of the ejaculatory latency in the masturbation and intravaginal, that accompanies of an improvement in the communication of couple, frequency of coitus and sexual satisfaction, and could be an effective option for the treatment of E.P.

Pages 83-84

Authors: Sora Yasri, Viroj Wiwanitkit

The spermatogenic transmission of infectious disease is an interesting consideration in reproductive medicine. The problem can be serious and classified as sexually transmitted infection. The concern is on the new emerging viral infections because there is usually little information on those new viruses. In this short article, the authors specially review and discuss on Spermatogenic transmission of Marbug and ebola virus.

253-259

Authors: Jillian L. Sia Aleli, Gliceria B. Ramos, Miriam P. de Vera

Objective To assess the protective potential of α-tocopherol on ethanol-induced dysmorphogenesis in 10.5 embryonic day (ED) mouse embryos. Methods Forty female mice were randomly assigned into control (CON), positive control (ETOH), low-, medium and high-α-tocopherol-supplemented-Ethanol groups (LTOC, MTOC, HTOC, respectively). CON received drinking water without ethanol, ETOH, LTOC, MTOC and HTOC groups received 20% ethanol in drinking water. The supplemented groups were given respective dosages of α-tocopherol, 0.410, 0.819 and 1.640 mg/g body weight, at 14 days before mating until the 9th day of gestation. The 10.5 ED embryos were assessed for embryo weight, head- and crown-rump length, and morphological scoring of brain and sensory vesicles, flexion and somites. The embryo yield was assessed by counting the number of full-term developed embryos from the bulging implantation sites while resorption was assessed by counting the bulging implantation sites but without formed embryos. Results The weight and head- and crown-rump length of the embryos from the α-tocopherol supplemented groups were comparable to the control. These were significantly higher than that of positive control (P < 0.05). Overall morphological scores of the hindbrain and sensory vesicles were significantly higher in the supplemented and control groups than that of the positive control (P < 0.05). The number of full-term developed embryos was neither affected by ethanol alone nor with supplementation with α-tocopherol. Resorption was significantly lower in the supplemented groups than that of positive control (P < 0.05). Conclusion The medium and high dosages of α-tocopherol exhibited a protective effect on ethanol-induced dysmorphogenesis.

264-268

Authors: Deepika Jaiswal, Sameer Trivedi, Neeraj K. Agrawal, Kiran Singh

Objective To analyze global DNA methylation along with DNA methyltransferases (DNMTs) expression at transcript level in patients with impaired spermatogenesis to dissect its role in pathophysiology of human male infertility. Method The content of Global methylated cytosine (mC) was determined using ELISA system (Imprint Methylated DNA Quantification Kit, Sigma–Aldrich) in 31 testicular biopsies showing impaired spermatogenesis and 8 with normal spermatogenesis. Real-time reverse transcription-polymerase chain reaction was done to analyze DNMTs (DNMT1, DNMT3A, DNMT3B and DNMT3L) mRNA levels in biopsies with different testicular phenotypes. Results There was significant increase in levels of global methylation in different impaired testicular phenotypes as compared to normal. Expression analysis revealed significantly increased expression of DNMT1 and its positive correlation with global DNA methylation. Conclusion In conclusion, increased levels of global methylation in impaired cases might be the one of the contributing factors for aberrant gene expression in infertile patients.

275-287

Authors: S. Thangapandiyan, S. Miltonprabu

Objective To explore the ameliorative potential of Epigallocatechin gallate (EGCG) by evaluating markers of oxidative stress, apoptosis, and inflammation and antioxidant competence in Fl intoxicated rats. Methods The animals were divided in to four groups that is control, EGCG alone, NaF, and EGCG with NaF. Group III animal were exposed to Fl as sodium Fluoride (NaF) (25 mg/kg BW) for 4 weeks. After the completion of the treatment, the testis tissues has been removed and used for the experimental observations. Results Pre-administration of EGCG to Fl intoxicated rats showed a significant normalization in the levels of steroidogenic enzymes, testosterone, sperm functions, oxidative stress markers and antioxidant status. The altered levels of proinflammatory cytokines and apoptotic markers were also relapsed in close proximity to control. In addition, EGCG significantly improved antioxidant status and reduced the oxidative stress and pathological changes in testes. The mRNA and protein analysis also substantiated that EGCG pre-treatment markedly enhanced the expression of Nrf2 and its target genes HO-1, NQO1 and γGCS and suppressed the expression of Keap1 in testis. Conclusion Altogether, our findings supports that EGCG attenuates Fl toxicity in testis through Nrf2 activation.

297-304

Authors: Gulfam Ahmad, Muhammad Arslan

Objective The present study was undertaken as a part of a continuing search for proteomic based approaches for the diagnosis of ovarian cancers. Methods The study comprised four groups of women with: i): malignant ovarian cysts, ii) benign ovarian cysts, iii): breast cancer (positive controls) and, iv) healthy females (negative controls). Serum and cystic fluids were processed for gel electrophoresis (SDS-PAGE). Serum and cystic follicular fluid estradiol and testosterone concentrations were measured by ELISA. Results Proteomics analysis revealed the presence of a uniquely expressed protein with MW of 35 kDa in the serum and cystic fluid of patients with malignant ovarian cysts. On the other hand a protein of 100 kDa was not expressed in malignant ovarian patients whereas it was differentially expressed in the cystic fluid and serum of other three groups. Although mean values of estradiol levels were discernibly higher in patients with benign ovarian cysts as compared to those with malignant ovarian cysts and of positive and negative controls, difference was not significant. Mean concentrations of estradiol were significantly higher in cystic fluid aspirated from benign cysts as compared to that from malignant cysts. Conclusions Taken together these data indicate a uniquely expressed protein of 35 kDa in patients with malignant ovarian cysts that may serve as a specific protein biomarker for the differential diagnosis of the ovarian cancer. However, a much larger sample of subjects is required to validate and confirm these findings.

309-312

Authors: N. Faria, J. Simões

Objective To determine the incidence of uterine torsion and their association with live births after vaginal delivery at pasture. Methods A total of 119 veterinary-assisted dystocia, occurred in Holstein-Friesian cows, were accessed between September 2012 and February 2013 from Azores islands (Portugal). The general health status of dam, cause of dystocia, obstetric treatment choice, number and viability of fetus were evaluated. Results The uterine torsion represented 24.4% (29/119) of total assisted dystocia. The general health status was normal or slowly affected, at delivery time, for 96.6% (28/29) of the cows with uterine torsion. Vaginal delivery after rolling cows, cesarean section or euthanasia were performed in 72.4% (21/29), 24.1% (7/29) and 3.5% (1/29) of dams with uterine torsion, respectively. Singletons were observed in 96.6% (28/29) and 86.7% (78/90) of cows with uterine torsion and remaining dystocia, respectively. After vaginal delivery, the occurrence of singleton live births was more probable to occur in cows with uterine torsion (17/21) than the remaining cows (37/70) of control group (odds ratio = 3.79; 95% interval of confidence from 1.16 to 12.41; P < 0.05). Conclusions A high frequency of uterine torsion was observed in Holstein-Friesian cows with normal or slowly affected general health status at delivery time at pasture. The singleton live births prevailed and, in general, their occurrence by vaginal delivery after uterus reposition were most likely to occur in cows with uterine torsion than dams presenting other dystocia.

318-322

Authors: J. Silva, D. Rocha, I. Cunha, L. Rui Sales, F. Neto, M.C. Fontes, J. Simões

Objective To evaluate/predict the offspring serum profile of antibodies against Porcine reproductive and respiratory syndrome virus (PRRSV) in an affected intensive herd, whereas sows are vaccinated, and determine the better sample time using pigs as sentinels. Methods Serum samples were collected from a total of 66 offspring pigs from a PRRSV endemic herd, whereas only sows were vaccinated. Six animals per grouped age were randomly selected with 0 (at perinatal time), 1, 2, 3, 6, 9, 12, 15, 18, 21 and 24 weeks of age. Individual and three pooled samples were tested by ELISA and PCR, respectively. Results The proportion of seropositive animals was 75.0% (18/24), 33.3% (6/18) and 95.8% (23/24) at farrowing (0–3 weeks), nursery (6–12 weeks) and growing/finishing (15–24 weeks) phases, respectively. It was 46 times more likely (P < 0.01) to observe seropositive pigs on the growing/finishing than on nursery phase. European PRSSV strain was detected on serum of 6, 9 and 15-weeks-old pigs. Polynomial fit degree 4 regression between sample-to-positive (S/P) ratios and age of pigs (r = 0.78; R2 = 0.60; P < 0.001) estimated a quickly decrease of maternal antibodies, above 0.4 S/P value (cut off), until the 6th week following by a progressive increment of humoral immunity until 21st week of age, when it declines. Conclusions The results indicate that the piglets can present viremia early at nursery, and the 18th – 21st weeks are the best time to sample offspring pigs, as sentinel, from PRRSV contaminated intensive herds.

334-336

Authors: Achour Radhouane, Ben Aissa Imen, Neji Khaled

Twin pregnancy consisting of a complete hydatidiform mole coexisting with a live fetus is a rare condition with an incidence of 1 in 22 000 to 1 in 100 000 pregnancies. Clinical information is limited and management is difficult due to the risk of pregnancy complications such as fetal death, vaginal bleeding, preeclampsia, hyperthyroidism, and the risk of persistent gestational trophoblastic disease. Thus, the report described about the change of size and implantation site of the molar mass coexisting with a live fetus is rare especially about sonographic findings such as echo patterns. Recently we experienced a case of complete hydatidiform mole with a healthy infant delivered at term.

Pages 169-177

Authors: Ansoumane Kourouma, Peng Duan, Hady Keita, Aidogie Osamuyimen, Suqin Qi, Chao Quan, Tingting Yu, Kedi Yang

Objective To explore the mechanism by which nonylphenol (NP) interferes with male infertility through evaluation of its effects on epididymal sperm of adult male rats. Methods Twenty four Sprague–Dawley (SD) rats were used as epididymal sperm donors. Previously rats were administrated with NP (0, 2, 10 and 50 mg/kg) body weight respectively in corn oil every forty-eight hours by intraperitoneal injection for 30 days. Computer assisted sperm analysis (CASA) was used to determine parameters of sperm. The sperm morphology examination was conducted with a high resolution microscope. Results Results indicated that exposure to NP has no effect on body weight, while testes weights were significantly decreased. Computer assisted sperm analysis (CASA) showed significant decline in the percentage of motile spermatozoa (P < 0.001), STR and LIN (P < 0.01), significant increase in ALH (P < 0.001), while significant decline in BCF (P < 0.001) respectively. Plasma LDH was significantly increased while; plasma γ-GT activity was significantly decreased. H2O2 production and malondialdehyde (MDA) were significantly increased. The Plasma CAT, GSH-Px and SOD activities were significantly decreased. Conclusions This concludes that NP leads oxidative stress in the epididymal sperm of rats. Moreover, NP can disrupt sperm motility and alterations in the sperm morphology.

Pages 185-191

Authors: Nishant Shinde, Akhilesh S. Chauhan, Sanjay K. Gupta, Surendra H. Bodakhe, Devi Prasad Pandey

Objective To evaluate the antifertility potential of curcumin and andrographolide combination in female rats by studying its effect on implantation and estrus cycle. Methods Female Sprague Dawley rats (180 ± 30 g) were used for study. They were divided into groups and were treated as per schedule. The antifertility effect of curcumin and andrographolide was evaluated by measuring their effect on implantation and estrus cycle of rats. Also their antigonadotropic and antiestrogenic effects were evaluated using suitable screening method. Results The combination significantly reduced the number of implants and the size of the litters in rats compared to the normal control group. The combination also significantly altered the durations of each phase of estrus cycle and synergized the effect to decrease the number of ovarian follicles. Conclusion The results indicate that the combination produces greater antifertility effects and thus can play a vital role in fertility control.

Pages 198-204

Authors: Abhinandan Ghosh, Kishalay Jana, Bhabani Prasad Pakhira, Adrija Tripathy, Debidas Ghosh

Objective To explore the anti-fertility efficacy of aqueous-ethanolic (1:1) extract of fruits of Terminalia chebula (T. chebula). Methods Aqueous-ethanolic (1:1) extract of fruit of T. chebula was administered orally at a dose of 60 mg/0.5 mL distilled water/day for 28 days. Different parameters were studied including body weight, relative weight of reproductive organ, sperm motility, sperm count, testicular cholesterol, plasma testosterone, testicular androgenic key enzymes such as Δ5, 3β-HSD and 17-β-HSD, bio-markers of oxidative stress, toxicity study and histological analysis of the tissues. Results The treated group showed a significant diminution in spermatogenic profile. On the other hand testicular cholesterol showed a significant elevation in T. chebula treated group and plasma testosterone was decreased significantly in comparison to control. The above said androgenic key enzymes were exerted a significant diminution in extract treated group. Anti-oxidative enzymes such as catalase and superoxide dismutase showed a significant reduction and a significant elevation in conjugated diene and Thiobarbituric acid reactive substance was noted in treated group. GOT and GPT study of liver and kidney showed a non-significant change which confirmed the non-toxic nature of T. chebula. Histological study of testis of treated group exhibited significant reduction in seminiferous tubular diameter. Conclusion The results of present experiment suggested that the aqueous-ethanolic (1:1) extract of fruit of T. chebula exerted a significant anti-spermatogenic effect in male rat.

Pages 216-220

Authors: Wei-Siong Lim, Mary-Shi-Ying Gan, Melissa-Hui-Ling Ong, Choy-Hoong Chew

Objective To investigate the effect of different concentrations of lauric acid on Intercellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1) expression in IFN-γ stimulated human monocytic THP-1 cell line. Methods THP-1 cell were cultured using Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum. THP-1 monocytes were firstly differentiated into macrophages by using phorbol-12-myristate-13-acetate. IFN-γ response test was performed and total cellular RNA was extracted using TRI Reagent® LS before q-RT-PCR was carried out. Subsequently, IFN-γ treated THP-1 macrophages were stimulated with increasing doses of lauric acid for another 24 h, before q-RT-PCR. Results The mRNA expression levels of ICAM-1 and VCAM-1 were normalized to β-actin and relatived to the untreated cells. The expressions of ICAM-1 and VCAM-1 were significantly induced in cells treated with 10 ng/mL of IFN-γ. This showed that IFN-γ could up-regulate inflammatory process and may cause atheroma formation. MTT assay was carried out to investigate the effect of lauric acid on undifferentiated and differentiated THP-1 cells. Although lauric acid did not have any significant impact on undifferentiated and differentiated THP-1 cell viability, the normalized fold expressions of ICAM-1 and VCAM-1 in IFN-γ-treated THP-1 macrophages were decreased significantly in a dose dependent manner with the presence of increasing doses of lauric acid. Conclusions This study successfully proved that lauric acid was able to antagonize the up-regulatory effect of IFN-γ on ICAM-1 and VCAM-1 expressions in THP-1 macrophages. This indicates that lauric acid may be an anti-inflammatory therapeutic and prophylaxis agent for atherosclerosis.

210-215

Authors: Ibukun P. Oyeyipo, Yinusa Raji, Adeyombo F. Bolarinwa

Objectives To evaluate the effects of inhibiting nitric oxide synthase as a means of intervention in nicotine-induced infertility in male rats. Methods Forty-eight male and thirty female Wistar rats (180–200 g) were randomly assigned to six groups and treated orally for 30 days with saline (control), nicotine (0.5 mg/kg, 1.0 mg/kg) with or without NG Nitro-l-Arginine Methyl Ester (l-NAME, 50 mg/kg). Treated male rats were cohabited with untreated females in ratio 1:2 for fertility studies. Sperm analysis was done by microscopy. Results There was a significant decrease in the epididymal sperm motility and count after nicotine treatment. However, the percentage of abnormality significantly increased in nicotine treatment groups. Fertility studies revealed that nicotine reduced libido in male rats and decreased litter weight and number delivered by the untreated female during the experiments. Co-treatment with l-NAME effectively reversed the nicotine-mediated alterations in the sperm functional parameters, fertility indexes and hormone when compared to nicotine only. Conclusion Taken together, the present data indicate the abilities of l-NAME to ameliorate nicotine-induced spermatotoxic effects in male rats via a mechanism dependent on the circulating testosterone level.

228-235

Authors: Boubakari Ibrahimou, Hamisu M. Salihu, Muktar H. Aliyu, Gary English, Getachew Dagne

Objective To assess whether conversion from twin to singleton pregnancy following the demise of a co-twin influences survival. Methods This retrospective study compared the risk for neonatal, post-neonatal and infant death for converted co-twins versus unconverted co-twins using the US matched multiple file dataset for the period 1995–2000. We also examined the same risks for converted versus same-quantile co-twins, hazard ratios (HR) and 95% confidence intervals (CI) were computed using Cox Proportional Hazards models. Results The risk for neonatal (HR = 0.18, 95% CI: 0.09–0.34 and HR = 0.69, 95% CI: 0.50–0.96) and infant death (HR = 0.22, 95% CI: 0.12–0.42 and HR = 0.57, 95% CI: 0.42–0.77) were significantly lower for converted twins than for unconverted twins and same-quantile twins, respectively. For black compared to white, the risk for post-neonatal death increased by 89% (HR = 1.89, 95% CI = 1.03, 3.48), and 79% (HR = 1.79, 95% CI = 1.53, 2.09) for converted vs. unconverted and converted vs. same-quantile, respectively. For converted black, the risk for neonatal death decreased by 17% (HR = 0.83, 95% CI = 0.73–0.93) as compared to unconverted. Conclusions Risks for all mortality types were lower among converted co-twins than their unconverted or same-quantile counterparts. The lower neonatal and higher post-neonatal mortality among black require future research.

242-243

Authors: Natalie K. Binder, Natalie J. Hannan, David K. Gardner

Objective To determine the efficacy of embryo outgrowth on fibronectin as a low cost, high throughput alternative to embryo transfer to model embryo attachment and the initial stages of implantation. Methods Following in vitro embryo culture, embryo quality was assessed via embryo transfer or embryo outgrowth with metabolic assessment. Results This study shows that blastocysts attach to fibronectin at the same rate that they implant in vivo, and that the carbohydrate utilisation of embryos that successfully outgrow is comparable to those that are able to develop into a fetus. Conclusions Embryo outgrowth is a suitable alternative endpoint to embryo transfer.

251-252

Authors: Achour Radhouane, Basly Mohamed, Ben Aissa Imen, Ferjaoui Aymen, Neji Khaled

Congenital cervical atresia and hypoplasia are rare abnormalities that generally require reconstructive or extirpative procedures to relieve outflow tract obstruction. Infertility is a common sequel, and only four previous pregnancies have been reported. We report a case of successful pregnancy after in-vitro fertilization in a 32-year-old patient with congenital cervical atresia diagnosed at the age of 28 years. She was referred to our unit and had a successful pregnancy after in-vitro fertilization. Caesarean section was performed at 38 weeks gestation. A healthy male baby weighing 3650 g was safely delivered.