Asian Pacific Journal of Reproduction

55-63

Authors: Hayder M. Al-kuraishy, Ali I. Al-Gareeb, Thabat J. Al-Maiahy

Objective: To explore the association between statin therapy and the risk of erectile dysfunction by literature review. Methods: We conducted diversities of search strategies including electronic database searches of MEDLINE, Scopus, Pubmed and Web of Science using MeSH terms, keywords and title words during the search. Reference lists of identified and public articles were reviewed. In addition, only English articles were considered and case reports were not concerned in the review. The key features of recognized applicable search studies were considered and the conclusions were summarized in a narrative review. Results: Different studies gave a consensus that erectile dysfunction was regarded as an early sign of silent cardiovascular disorder and hidden atherosclerosis. Different studies reported that statins might induce erectile dysfunction through induction of peripheral neuropathy, cognitive deficits, and reduction of circulating testosterone. However, most of recent studies illustrated that statins led to a significant improvement in erectile function and sexual health in men with age over forty years. Atorvastatin advanced endothelial nitric oxide concentrations through activation and upregulation of endothelial nitric oxide synthase and rescued phosphodiesterase-5 inhibitors non-responders since nitric oxide and cyclic guanosine monophosphate increased penile blood flow and improved erectile function. Conclusions: According to the assorted view of preponderance, statins improved erectile dysfunction is more dominant than statins induced erectile dysfunction. Therefore, statins regardless of its property improve erectile dysfunction through amelioration of penile endothelial dysfunction, and penile neuronal reflexes that are inter-related during sexual excitation and penile erection.

70-76

Authors: Rini Widyastuti, Arief Boediono, Mas Rizky Anggun Adipurna Syamsunarno, Mohammad Ghozali, Mulyanusa Amarullah Ritonga, Alkaustariyah Lubis, Sondi Robianto, Jaqueline Sudiman

Objective: To assess the effect of short-term gavage of ethanolic extract of Imperata cylindrica L root on the ovarian activity and estrus behavior of female mice. Methods: Eighteen virgin female ddY mice, 8 to 10 weeks of age, weighing 22–25 g with regular estrus cycle, were divided into three groups. Group 1 received 0.5% carboxymethylcellulose, whereas Groups 2 and 3 received the ethanolic extract of Imperata cylindrica L root at 90 and 115 mg/kg body weight (b.w.) per day by gavage for 20 days, respectively. All of the groups were checked before 9 a.m. daily for vaginal cytology to determine the estrus phase. On day 21, the mice were sacrificed to collect serum samples to quantify the concentrations of reproductive hormones using enzyme-linked immunosorbent assay and to determine changes in the reproductive organs based on their reproductive organ weight, histomorphology, and histomorphometry of ovarium and uterus. Results: The reproductive organ weight in the treatment groups was similar compared with that in the control group. The 90 mg/kg b.w. treatment group showed an increase in corpus luteum number when compared with the control group, with few degenerated follicles and diminished oocytes. Moreover, the 115 mg/kg b.w. treatment group showed fewer primordial and primary follicles and an increase in corpus luteum number and a prolonged diestrus phase compared to the control and 90 mg/kg b.w. treatment groups. The histomorphology examination of the uterus showed that the thickness of myometrium and epithelium in the treated animals was similar to the control group. In addition, there was a significant decrease in follicle-stimulating hormone level in the 115 mg/kg b.w. treatment group (P<0.05). Conclusions: Short-term gavage of ethanolic extract of Imperata cylindrica L root reduces the follicle-stimulating hormone serum level and folliculogenesis.

83-88

Authors: Reza Kheirandish, Ladan Emadi, Baharak Akhtardanesh, Shahrzad Azizi, Masoud Imani, Fatemeh Mahmoodabadi, Fereshteh Irani, Homa Shokrizadeh

Objective: To investigate the hormonal, histopathological toxicity and sperm quality of ciprofloxacin in male genital system in the rabbit model. Methods: Twenty adult New Zealand male rabbits were randomly divided into the treatment and control groups, and respectively received 30 mg/kg/day ciprofloxacin and normal saline intraperitoneally for 14 days. Blood samples and testicular tissues were taken for testosterone by enzyme linked immunosorbent assay method, and measurement of histopathologic and histomorphometric investigations was made on the 14th day of the experiment and 56 days after the last dose of ciprofloxacin. Additionally, epididymis sperm sample was collected for quality analysis. Results: On day 14 of the experiment, histopathologic examination revealed severe degenerative changes in the epithelium of seminiferous tubules and loss of all germ cells in most tubes in the treatment group. In the histomorphometric study, significant reductions in the seminiferous tubules diameter, epithelium height, and Johnsen’ score were observed. On day 56 after the last dose of ciprofloxacin, the seminiferous tubules revealed regeneration in the treatment group and no significant difference was observed in the spermatogenesis parameters except epithelial height parameter between the control group and the treatment group. All sperm quality parameters were significantly decreased on day 56 after the last dose of ciprofloxacin. Testosterone levels did not significantly change during the follow-ups period. Conclusions: There is potential reversible testicular toxicity for ciprofloxacin in the rabbit model in according to relative regeneration of spermatogenic epithelium after 56 days of last dose of ciprofloxacin. Sperm quality will be improved with a delay after complete regeneration of seminiferous tubules.

96-103

Authors: Ahmed R. M. El-Khawagah, Zaher M. Rawash, Diya A. El-Badry, Mohamed M. M. Kandiel

Objective: To evaluate the cryoprotective effect of butylated hydroxytoluene on buck frozen semen. Methods: Semen was collected from Boer (n=6) and Zaraibi (n=6) bucks by electroejaculator for 5 weeks. Semen aliquots were diluted at 38 ℃ in Tris-buffer with egg yolk 15.0% (vol/vol) (Tris- egg yolk extender) or soya lecithin 2.5% (weight/vol) (Tris-soya lecithin extender) supplemented with butylated hydroxytoluene at 0.0 (as the control), 0.5, 1.0, 2.0 and 4.0 mM. Post-thawing motility (at 400× magnification), plasma (hypo-osmotic swelling test), acrosome (Trypan blue/Giemsa dual staining) membranes, DNA (comet assay), and lipid peroxidation (by malondialdehyde concentration) were assessed. Results: Spermatozoa motility was enhanced by butylated hydroxytoluene in Tris-soya lecithin extender at 0.5 mM in the two breeds, and in Tris-egg yolk extender at 1.0 mM in Boer and at 2.0 mM in Zaraibi bucks for up to 3 h post-thawing. Plasma and acrosome membranes and DNA integrity of the two breeds were maximally high with butylated hydroxytoluene at 1.0- 2.0 mM in Tris-egg yolk extender and at 0.5-1.0 mM in Tris-soya lecithin extender. Lipid peroxidation was minimal with butylated hydroxytoluene at 1.0-2.0 mM in Tris-egg yolk and soya lecithin extenders in the two breeds. Butylated hydroxytoluene at 4.0 mM deteriorated spermatozoa motility, and plasma and acrosome membranes. Conclusions: The consequence of butylated hydroxytoluene on buck frozen-thawed spermatozoa varies with the levels of supplementation, buck breed, and phospholipid source in the extender. Semen parameters of Boer buck are better in their response to butylated hydroxytoluene than Zaraibi buck. Butylated hydroxytoluene at 1.0 and 2.0 mM in Tris-egg yolk extender, and at 0.5 mM in Tris-soya lecithin extender represents the best concentrations and profitably improves the semen quality of buck semen.

109-110

Authors: Menizibeya O. Welcome

No

255-260

Authors: Nandini Sharma, Shiva Pratap Singh, Alok Bharadwaj, Ramachandran Natesan

Early diagnosis of pregnancy plays an important role to minimize reproductive losses in farm animals. There are several methods for pregnancy diagnosis like profiling of reproductive hormones (such as progesterone and estrone sulfate), but sometimes they provide false-positive results. Embryo specific pregnancy markers, which delineate the presence and viability of the embryo, are considered as perfect for pregnancy determination. Pregnancy-associated glycoproteins are distinguished as the best indicator for the determination of early pregnancy, fetal number, and birth weight of kids. Pregnancy-associated glycoproteins are structurally correlated to aspartic proteinase and are communicated in the external epithelial cell layer of the placenta. They have been found to share about half amino acid sequence identity with pepsinogen, pepsin, cathepsin D and E. Dislike different individuals from aspartic proteinase family, numerous pregnancy-associated glycoproteins appear to be latent compound as a result of amino acid substitutions in and around the catalytic site. This review is to discuss the scope and prospects of pregnancy-associated glycoproteins as a pregnancy marker in farm animals, more specifically in goats.

267-274

Authors: Dibyendu Ray, Sunidhi Roy, Pradip Panda, Partha Nandi, Sandip Mukherjee, Subrata Ghosh

Objective: To investigate the effect of Camellia (C.) sinensis in mitigating oxidative damage and reproductive toxicity in testis induced by sodium fluoride in a rat model. Methods: Twenty-four adult male Wister rats were divided into 4 groups, with 6 rats in each group. Group 1 orally received distilled water (1 mL/100 g body weight) daily and served as the control group, while group 2 received drinking water with 100 ppm sodium fluoride per day for 21 consecutive days, group 3 was administered with only C. sinensis extract by gavage at a dose of 100 mg/kg body weight and group 4 received drinking water with 100 ppm sodium fluoride and 100 mg/kg body weight C. sinensis leaf extract per day for 21 consecutive days. At the end of the treatment, the rats were sacrificed under light ether anesthesia. The gonado-somatic index, sperm count and motility, serum level of luteinizing hormone and testosterone were assayed. Lipid peroxidation [malondialdehyde (MDA) level], nitric oxide (NO) production, and activities of antioxidant enzymes - superoxide dismutase (SOD), catalase, and reduced glutathione level (GSH) were also analysed. Results: Sodium fluoride treatment significantly decreased gonadosomatic index, sperm count and motility as well as the serum level of luteinizing hormone and testosterone (P<0.05). The histological examination of testes revealed atrophy and degenerative changes in several seminiferous tubules, along with enhanced interstitial space and a reduced number of Leydig cells. There was a highly significant increase in NO and MDA production (P<0.05), while SOD, catalase activities and GSH level decreased significantly (P<0.05). However, C. sinensis significantly restored testicular weight, sperm parameter, hormonal level (P<0.05), and also reversed MDA and NO generation and antioxidant enzymes activities in the testicular tissue (P<0.05). Conclusions: C. sinensis may have an ameliorative role against sodium fluoride-induced oxidative damage in the testis probably because of its antioxidant property.

283-290

Authors: Wahab Adekunle Oyeyemi, Oore-oluwapo Ololade Daramola, Adeniran Oluwadamilare Akinola, Adeoye Oyewole Idris, Ikponmwosa Aikpitanyi

Objective: To investigate the hepatic and reproductive toxicity of dichlorvos and lead acetate on male Wistar rats. Methods: Fifteen adult male Wistar rats (170-190 g) were randomly divided into three groups, with 5 rats in each group. Group 1 received 0.5 mL distilled water orally and served as the control group, while groups 2 and 3 were orally treated with 2 mg/kg body weight (b.w.) dichlorvos and 10 mg/kg b.w. lead acetate, respectively, for 55 days. Epididymal sperm, serum follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone concentrations, testicular 17β-hydroxysteroid dehydrogenase activity (17β-HSD), androgen receptor expression, aspartate aminotransferase (AST), alanine aminotransferase (ALT), testicular oxidant and antioxidant enzymes were evaluated with standard methods. Results: Sperm count, motility, morphology, FSH, LH, testosterone levels, 17β-HSD, androgen receptor expression, and catalase activity were significantly reduced in the dichlorvos and lead acetate treated groups as compared with the control group (P<0.05). The liver AST, ALT activities and malondialdehyde concentration were significantly increased in the dichlorvos and lead acetate treated groups as compared with the control group (P<0.05). Conclusions: The reproductive and hepatic toxicity activities of dichlorvos and lead acetate in male Wistar rats are similar.

298-304

Authors: Ali Talebi, Shadan Navid, Maryam Borhani-Haghighi, Yumi Hoshino, Mehdi Abbasi, Zahra Khosravizadeh

Objective: To investigate the effects of soft agar on in-vitro proliferation of neonate mouse spermatogonial stem cells cocultured with Sertoli cells. Methods: Tissues of neonate NMRI male mice testes were used for harvesting spermatogonial stem cells and Sertoli cells. After cell harvest, flow cytometry using promyelocytic leukemia zinc-finger (PLZF) protein antibody was used to assess the purity of the cells. The isolated testicular cells were cultured in the absence (the control group) or presence of soft agar-coated dishes (the experimental group) supplemented with leukemia inhibitory factor and glia cell line–derived neurotrophic factor for two weeks. Alkaline phosphatase activity was assessed in the colonies formed after two weeks of culture by alkaline phosphatase staining. On day 14 of culture, the expression levels of DNA-binding protein inhibitor (ID-4) and PLZF genes in the undifferentiated cells were evaluated by the detection of PLZF protein antibody using real-time PCR and immunocytochemistry techniques. The number and diameter of the colonies of spermatogonial stem cells were assessed by ImageJ software. Results: In the experimental group, the number and the diameter of colonies significantly increased as compared with those in the control group (P<0.05, P<0.01, respectively). In addition, the level of expression of ID-4 and PLZF genes in the undifferentiated cells significantly increased in the experimental group as compared with the control group (P<0.01). However, the expression of tyrosineprotein kinase kit (c-kit) gene in differentiated cells decreased in the experimental group as compared with the control group, but there was no significant difference between the two groups. Conclusions: Spermatogonial stem cells can be efficiently proliferated by culturing the stem cells in soft agar-coated dishes. The new protocol used in this study can be a valuable method for future studies.

211-214

Authors: Roland Eghoghosoa Akhigbe, Moses Agbomhere Hamed

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may have a ripple effect that puts men at a risk of infertility. This article reviews the possible link between SARS-CoV-2 infection and male reproduction following speculations that the singlestranded RNA viruses could directly invade the testes. SARS-CoV-2 enters the human lung cells via angiotensin converting enzyme 2 (ACE2). ACEs, its products, angiotensin-(1-7), and its receptor, MAS receptor, are expressed in the testes. Although the binding of SAR-CoV-2 to ACE2 could lead to excess angiotensin Ⅱ with possible enhanced inflammation, angiotensin Ⅱ could also promote sperm motility. In addition, the pathophysiology of SAR-CoV-2, especially in relation to male fertility, is yet to be fully understood; the suppression of androgen observed in COVID-19 infected men calls for the need for andrological assessment in infected male.

223-229

Authors: Roghayeh Ghomi, Parvaneh Vasli, Meimanat Hosseini

Objective: To investigate predictors of caring behaviors of mothers of premature infants based on the health belief model. Methods: This cross-sectional study was conducted by using the structural equation modeling on 168 mothers of premature infants, who were selected by convenience sampling method from October 2017 to February 2018 in Iran. Data were collected by using a standard scale. Validity and reliability of all data collection tools were approved. Data were analyzed by using SPSS V.16 and Mplus6 software. Results: The structural equation modeling of the initial health belief model did not have a good fit, but the fitness of model 2 obtaining from the modified initial model was confirmed by changes in locations of constructs. None of constructs of model 2 had a significant positive association with the caring behavior of mothers of premature infants and only 2.8% of variance of caring behaviors in mothers could be predicted by the sum of variables of demographic characteristics and the modified health belief model constructs. Conclusions: Given that the findings do not approve the use of the health belief model in predicting determinants of caring behavior of mothers of premature infants, it is suggested to apply this model to investigate the effect of educational intervention based on the health belief model on the caring behavior of mothers.

239-246

Authors: Kambulu Lukusa, John Kabuba

Objective: To evaluate the effects of semen collection methods and different cooling rates on cooled and frozen-thawed Saanen goat semen. Methods: Twenty bucks were divided into two groups of 10 animals per group based on semen collection methods: artificial vagina and electro-ejaculator. Samples from each collection method were pooled and diluted with Tris-based extender. The pooled semen was divided into two cooling methods: slow cooling and fast cooling. Sperm motility and velocity, sperm plasma membrane integrity, sperm viability, mitochondrial membrane potential and DNA integrity were evaluated before dilution, immediately after cooling at 4 ℃ and at 24 h after freezing by using computer assisted sperm analyser method and epifluorescence microscope. Results: Mean values for total motility, rapid-speed and progressive motile spermatozoa were significantly higher in semen collected with artificial vagina than electro-ejaculator (P<0.001). Slow cooling resulted in higher percentages of total motile, rapid-speed and progressive motile spermatozoa as compared to fast cooling (P<0.05). The post-thaw percentage of rapid-speed spermatozoa collected by artificial vagina in slow cooled sperm was significantly higher as compared to electro-ejaculator method (P<0.05). The mean values of curvilinear velocity and straight-line velocity were significantly higher in slow cooled semen obtained by artificial vagina (P<0.001). Similarly, the combination of artificial vagina and slow cooling resulted in significantly higher post-thaw percentages of straight-line velocity, average-path velocity, sperm plasma membrane integrity, viability and acrosome integrity (P<0.01). No significant differences were observed in slow cooling between artificial vagina and electro-ejaculator in terms of post-thaw sperm mitochondrial membrane potential and DNA integrity (P>0.05). Conclusions: The use of artificial vagina and slow cooling rate in cryopreservation protocol improves the cooled and post-thaw sperm quality of Saanen buck semen.

159-165

Authors: Ayoob Rostamzadeh, Reza Ahmadi, Mahdi Heydari, Amir Raoofi

Nitric oxide (NO), a member of the reactive nitrogen species family, plays a role in several physiologic processes, including vasculogenesis and angiogenesis, growth and puberty, and senescence and apoptosis. NO plays an important role in the production of ovarian steroids, ovulation, and follicular apoptosis. In other words, increased activity of nitric oxide synthase (NOS) leads to an increased amount of NO, which triggers production of prostaglandins and inflammatory cascades which facilitate follicular rupture and atresia. NO concentration elevation inhibits steroid synthesis in luteal and granulosa cells. Since NO is a major paracrine mediator of various biological processes, as well as a key factor in both the reproductive cycle and embryo implantation, oversynthesis of NO in the uterus results in toxicity and inflammation in epithelial cells and immunorejection of implantation. In the male physiological system, NO synthesized by NOS plays a major role in erectile function and androgen secretion, as well as semen parameters, and oocyte junction to the sperm. Furthermore, this supposedly simple molecule is involved in a number of other functions, such as germ cell evolution, connections between sertoli cells and germ cells in the blood-testis barrier, homodynamic contraction, and germ cell apoptosis. Moreover, NO is considered a key factor in male fertility due to its widespread distribution in both normal and diseased testis tissue. The difference of expression level of NOS in normal and pathological states is a probable cause of fertility destructive processes.

174-181

Authors: Degemu Sahlu, Negussie Deyessa, Naod Firdu, Sahle Asfaw

Objective: To determine the association between low birth weight and household food insecurity at government hospitals in Addis Ababa, Ethiopia. Methods: A case control study was carried out on 468 mothers with term neonates from February 1, 2017 to May 15, 2017. The cases were women who gave term babies weighing less than 2 500 g and the controls were those having 2 500 g or above. In the included hospitals, choices of cases were done as the cases found and the next three eligible newborns in the maternity room were the controls. Data were collected by using pretested and structured questionnaire. Standard beam balance was used to measure the neonatal weight by trained midwifery. The data were entered into a computer using Epi- Data 3.1 and exported to Stata version 14 for data management and analysis. Results: Mothers having food insecurity [adjusted odd ratio (AOR) 3.58; 95% confidence interval (CI) (1.79-7.16)], midupper arm circumference [AOR 7.70; 95% CI (4.39-13.60)], hypertension [AOR 4.81; 95% CI (2.33-9.93)], and early age [AOR 3.88; 95% CI (1.35-11.15)] showed statistically significant association with low birth weight. Conclusions: Household food insecurity, hypertension, midupper arm circumference and early age in women were significant predictors of low birth weight. The provision of adequate nutrient for pregnant mothers having household food insecurity should be assured in order to prevent adverse birth weight outcomes.

192-196

Authors: Pribakti Budinurdjaja, I Wayan Arsana Wiyasa, Ika Kustiyah Oktaviyanti, Djanggan Sargowo

Objective: To investigate whether administration of genistein can improve hormonal changes (estradiol and follicle stimulating hormone (FSH)] , heat shock protein 70 (Hsp70) expression, and thickness of vaginal epithelial cells in a rat model of vaginal atrophy. Methods: Twenty-five multiparous female rats were divided into five groups, namely the control group (without ovariectomy), the ovariectomy only group, and the ovariectomy groups 1, 2 and 3 receiving genistein at the doses of 0.045, 0.090 and 0.180 mg/kg body weight/day, respectively. Estradiol, FSH, and Hsp70 expression were analyzed by using the immunoassay technique. Analysis of the thickness of the vaginal epithelium was performed by histology. Results: Ovariectomy significantly decreased estradiol levels compared to the control group (P<0.05). All doses of genistein significantly increased levels of estradiol in rats with vaginal atrophy compared to the ovariectomy only group (P<0.05). Ovariectomy significantly increased FSH levels compared to the control group (P<0.05). All three doses of genistein restored FSH levels comparable to those in the control group (P>0.05). Ovariectomy significantly increased parabasal cell Hsp70 expression compared to the control group (P<0.05). Of all the genistein doses, only the dose at 0.045 mg/kg body weight/day restored the expression of Hsp70 to levels in the control group (P>0.05). Conclusions: Genistein is able to increase the thickness of the vaginal epithelium through hormone modulation and cellular stress suppression. Genistein is beneficial in the form of a herbal or alternative food for improvement of vaginal atrophy due to menopause.

204-210

Authors: Elshymaa A. Abdelnaby

Objective: To assess blood flow parameters as well as Doppler indices at the first six months of pregnancy in buffalo heifers. Methods: A total of 15 healthy, cycling, buffalo heifers were examined twice per month. Examination of Doppler ultrasonography started from the first month till the sixth months of pregnancy. All animals were subjected to transrectal Doppler ultrasonography to assess ovarian and uterine blood flow. Resistance index, pulsatility index, peak systolic velocity, end diastolic velocity, and blood flow rate were measured of both ipsilateral and contralateral to the fetus side. Results: The resistance index was positively correlated with the pulsatility index (r=0.62, P<0.01) but negatively correlated with all other parameters. For both ovarian and uterine arteries ipsilateral and contralateral to the fetus, there was a reduction in the resistance index, with a significant increase (P<0.01) in peak systolic and end diastolic velocities in both ovarian and uterine arteries. The peak systolic and end diastolic velocities of the ipsilateral ovarian and uterine arteries increased linearly till the six months of pregnancy with increased growth demands of the fetus. The same for the uterine branch of the ovarian artery was done in the middle uterine artery and umbilical artery ipsilateral to the fetus side. Conclusions: This study provides reference data of the hemodynamic changes in both ovarian and uterine arteries that could be a valuable tool to evaluate all hemodynamic changes in the developing placenta/fetus. Transrectal Doppler ultrasonography proves to be a useful non-invasive method to assess utero-ovarian blood flow during pregnancy.

1-8

Authors: Thabat J. Al-Maiahy, Ali I. Al-Gareeb, Hayder M. Al-kuraishy

Objective: To find the association between testosterone serum levels and severity of late onset pre-eclampsia. Methods: This case-control study involved 34 patients with preeclampsia and 24 healthy control pregnant women matched for gestational and maternal age. The recruited pregnant women were divided into two groups: the pre-eclampsia group (34 pregnant women with pre-eclampsia) and the control group (24 healthy pregnant women). Lipid profile, blood urea, serum creatinine, proteinuria, total serum testosterone, sex hormone binding globulin and free androgen index were evaluated. Moreover, body mass index and blood pressure profile were measured. Results: There were high systolic and diastolic blood pressures in the pregnant women with late-onset pre-eclampsia when compared with healthy pregnant women (P<0.01). Total serum testosterone was higher in women with pre-eclampsia compared with healthy pregnant women (P=0.001). The free androgen index was higher in women with pre-eclampsia compared with healthy pregnant women (P=0.001). Sex hormone binding globulin level was low in women with pre-eclampsia compared with healthy pregnant women (P=0.001). High total serum testosterone was significantly correlated with all measured variables (P=0.001), except for body mass index and pulse pressure (both P=0.070). Smoking habit was low in those patients compared with healthy pregnant women. Meanwhile, total serum testosterone serum level was significantly correlated with number of cesarean sections (r=0.86, P<0.01) and nulliparty (r=0.56, P<0.01). Conclusions: Late onset pre-eclampsia in pregnant women is associated with high serum levels of total serum testosterone, free androgen index, low sex hormone binding globulin, low smoking habit with positive history for nulliparity and caesarean sections that are correlated with high blood pressure profiles. Therefore, high total serum testosterone which is correlated with most of risk factors of late-onset pre-eclampsia is regarded as a proxy biomarker reflecting the severity of late onset pre-eclampsia.

16-21

Authors: Dono Indarto, Budiyanti Wiboworini, Amelya A. Ayusari, Arisanty N. Restuti, Isnar N. Alfiyah, Aniki Puspita, Yohanes C. Wibowo, Yoga M. Pratama

Objective: To assess the association between genetic variants of transferrin receptor 2 (TFR2) exon 4 and anemia status and to describe the expression levels of several cytokines, hepcidin, soluble transferrin receptor and erythropoietin. Methods: Institutional based comparative study was done randomly to recruit 106 pregnant women who attended antenatal care in three different health centers in Boyolali Regency, Central Java from May 2015 to September 2015. DNA was extracted from peripheral blood samples of selected pregnant women and sequencing was done for TFR2 exon 4. Furthermore, enzyme-linked immunosorbent assay was conducted to measure the expression levels of interleukin 6, interleukin 4, transforming growth factor β and iron-metabolism related proteins such as hepcidin, soluble transferrin receptor, and erythropoietin. Gene alignment was performed by using a CLUSTAL W program. Collected data were analyzed statistically by using parametric and nonparametric tests with Statistical Product and Service Solutions (SPSS) 20.0 for Windows. Results: Three novel genetic variants from TFR2 exon 4 (position 603, 605 and 606) were associated with anemia status. Moreover, the expression levels of interleukin 6, interleukin 4, transforming growth factor β and erythropoietin were higher in anemic pregnant women than those of nonanemic pregnant women but only erythropoietin level reached statistical significance. These results were followed by decreases of hepcidin and soluble transferrin receptor levels. Conclusions: Various factors contribute to anemia prevalence among pregnant women in Boyoali Regency, Central Java, Indonesia. Our novel findings showed that TFR2 exon 4 has 3 mutational sites in position 603, 605 and 606. These novel genetic variants may provide a new insight into the role of TFR2 in anemia.

31-36

Authors: Mandeep Kaur, Avtar Chand Rana, Sunil Kumar

Objective: To assess the estrogenic activity of hydroalcoholic extract of Clitoria (C.)ternatea leaves in female Wistar rats. Methods: Hydroalcoholic extract of C. ternatea leaves prepared by using cold maceration method was tested for estrogenic activity. An acute toxicity study was conducted to estimate the safe dose using OECD 423 guidelines. For estrogenic activity, ovariectomized female rats were divided into four groups, with 6 rats in each group. The control and standard groups were administered with 1% carboxymethyl cellulose orally and estradiol valerate at 1 μg/rat/day subcutaneously, respectively. The third group was administered with hydroalcoholic extract of C. ternatea at the dose 500 mg/kg body weight orally and the fourth group was administered with hydroalcoholic extract of C. ternatea at the dose 500 mg/kg body weight orally along with estradiol valerate at dose 1 μg/rat/day subcutaneously. All treatments lasted for 7 consecutive days and estrogenic activity was assessed by observing vaginal cornfication. On day 8, all animals were sacrificed and uterine horns were dissected out. Utrine weight was measured and blood serum was further processed for the estimation of biochemical parameters like cholesterol, total proteins, alkaline phosphatase and estrogen by autoanylser. Histological studies of uterus were also carried out. Results: Acute toxicity studies indicated the hydroalcoholic extract of C. ternatea leave was found to be safe up to the dose level of 2 000 mg/kg. Oral administration of C. ternatea extract at the dose 500 mg/kg body weight and and estradiol valerate (1 μg/rat/day) caused morphological changes i.e. increase in uterine weight, vaginal opening and cornification of cells; biochemical changes i.e. increase in cholesterol, total protein, alkaline phosphatase and estrogen contents; histological changes i.e. increase in uterine diameter, thickness and height of endometrium. Simultaneous administration of C. ternatea extract with estradiol valerate showed a synergistic effect. Histological investigations further confirmed the strong estrogenic nature of C. ternatea extract. Conclusions: C. ternatea extract (500 mg/kg) showed a significant estrogenic activity which is also supported by biochemical and histological studies. So, on the basis of these findings, it can be concluded that C. ternatea can be used as an alternative to synthetic oral contraceptives.

44-48

Authors: Seyed Morteza Aghamiri, Mohammad Rahim Ahmadi, Masoud Haghkhah, Abdollah Derakhshandeh

Objective: To investigate the bacterial infections in repeat breeder dairy cows and to evaluate the treatment effects of hyperimmune serum against two main endometritis bacteria, Escherichia (E.) coli and Trueperella pyogenes. Methods: A total of 29 Holstein multiparous cows with three or more unsuccessful artificial inseminations were carried out and examined to confirm the absence of reproductive tract abnormality and vaginal discharges. Uterine lavage was performed to collect uterine samples for bacterial and fungal cultures. In addition, polymerase chain reaction (PCR) was done to detect Trueperella pyogenes, E. coli, Fusobacterium necrophorum, and Prevotella melaninogenicus. The cytological study was done on cervical mucus. The hyperimmune serums produced against Trueperella pyogenes and E. coli were infused into the uterus of repeat breeder cows and two doses of prostaglandin F2α were administrated intramuscularly within 14 days' interval. Results: There were 10 positive samples in the bacterial culture with 19 isolations and no growth of the main causative bacteria of endometritis. In the PCR method, no Trueperella pyogenes, Fusobacterium necrophorum, and Prevotella melaninogenicus were found. However, 11 positive samples of E. coli were identified by PCR. Cladosporium was detected in one case of repeat breeder cows. The median (interquartile range) of neutrophils in vaginal discharge was 12 (22.5). Eighteen from twenty-nine (62.1%) repeat breeder and eight from eleven (72%) cows with E. coli infections in PCR were made pregnant in the first artificial insemination after treatment by intrauterine infusion of hyperimmune serum and prostaglandin F2α. Conclusions: Intrauterine administration of hyperimmune serum could be an alternative to antibiotics for the treatment of repeat breeder cows.