NaCl and cervical mucus

 rogen level and cervical mucus of Timor hind (Rusa timorensis) after mineral block supplementation during estrous cycle Daud Samsudewa1 , Enny Tantini Setiatin1 , Yon Supri Ondho1 , Isroli, Dinda Ayu Lestari 1 1 Faculty of Animal and Agricultural Science, Diponegoro University, Semarang, Indonesia * Corresponding author: daudreproduksi@gmail.com Abstract. The aim of this research was to observe the effect of mineral block containing Selenium, Magnesium and Zinc to the length of estrous of Timor hind based on estrogen level and cervical mucus (potential hydrogen (pH)) and natrium chloride (NaCl)). The materials used were ten Timor hinds with range of Body Condition Score (BCS) 2.75 ± 0.25 with first parity. Timor hinds were grouping into 2 groups, it was T0 = without mineral block supplementation and T1 = with mineral block supplementation for 8 weeks. After treatment, the estrous cycle of Timor hind was synchronized using vaginal sponge that contained 20 mg medroxy progesterone acetate (MPA) for 16 days. Cervical mucus and blood were collected at 0, 24, 72, 84, 96, 108, 120 and 144 hours after releasing of vaginal sponge. The cervical mucus was measured for pH and percentage of NaCl. The estrogen level was analysed by radio immunoassay (RIA). Data were analysed using non parametric statistics Mann-Whitney U test. The result showed significantly different was found on 120 and 144 hours of pH of cervical mucus. pH of cervical mucus on 120 and 144 hours were T0 = 8.46 and T1 = 8.30; T0 = 8.46 and T1 = 8.28, respectively. NaCl level on 72, 84, 96 and 108 hours were also significantly different in cervical mucus and estrogen level. The NaCl level of cervical mucus for 72, 84, 96 and 108 hours were T0 = 0.00181% and T1 = 0.00236%; T0 = 0.00179% and T1 = 0.00227%; T0 = 0.00178% and T1 = 0.00202%; T0 = 0.00156% and T1 = 0.00177%, respectively. The estrogen level for 72, 84, 96 and 108 hours were T0 = 8.21 pg/mL and T1 = 12,90 pg/mL; T0 = 7.54 pg/mL and T1 = 12.33 pg/mL; T0 = 4.56 pg/mL and T1 = 12.09 pg/mL; T0 = 3.76 pg/mL and T1 = 10.55 pg/mL, respectively. Estrous length of Timor hinds in group T0 and T1 was 48 and 84 hours, respectively. In conclusion, mineral block supplementation leads to longer estrous in Timor deer than those not given supplementation

A Study of Chloride Levels in Cervical Mucosa and Its Correlation with Transvaginal Sonography Dr. Rajani Shrivastava1 , Dr. Preeti Maheshwari2 , Dr. S. Sapre3 1Consultant Gynecologist, Arogyadham Hospital & Research Centre, Gwalior, Madhya Pradesh, India 2Consultant Gynecologist, Indore, Madhya Pradesh, India 3Ex- Obs & Gynic, Department of Obs. & Gynecology, GR Medical College, Gwalior, Madhya Pradesh, India Arogyadham Hospital & Research Centre, Gwalior, Madhya Pradesh, India *Corresponding author Dr. Rajani Shrivastava Email: shrivastavadrrajani@gmail.com Abstract: Ovarian function can be evaluated by various procedures. Among various constituents of cervical mucus, Sodium Chloride (NaCl) is the major electrolyte responsible for positive fern test(A positive Fern Test indicates presence of amniotic fluid in vagina. This occurs when membranes rupture. A negative Fern Test indicates absence of amniotic fluid in vagina.) at the time of ovulation. The Aim was to evaluate variations in sodium chloride level in cervical mucus and establish a relationship between peak chloride level in cervical mucus by ovulatory& ovulation by premenstrual transvaginal sonography in both infertile and fertile patients. The Study was conducted upon 100 women of reproductive age group with no obvious gynaecological or obstetric disease. There were two groups group I of 50 infertile women and group II of 50 fertile women. History, physicalsystemic-gynecological examinations, blood-urine-semen tests, Kahn's test and VDRL test were carried out before enrolment. Each patient was called thrice for TVS and cervical mucus examination. In Results In preovulatory phase, chloride level of all patients were =<0.5%. In periovulatory phase, 40% of both cases showed 0.7%, 6% showed 0.9% in infertile whereas 20% showed 0.9% chloride level in fertile cases. In postovulatory phase, chloride level of all patients were =<0.5%. According to endometrial pattern, 72% infertile and 92% fertile cases showed proliferative endometrium in concordance with follicular size of > 16 mm. 68% infertile and 92% fertile cases showed secretory endometrium which was taken as confirmative of ovulation. For chloride estimation, chloride level estimation detected ovulation in only 72% infertile cases & all fertile cases while cycle was ovulatory in 68%infertile cases & 92% fertile cases according to TVS. In Discussion the Chloride levels in ovulatory cycle were found to be <0.5% in 96% of cases during preovulatory phase, >=0.5% in 72% infertile and all of fertile cases during periovulatory phase, <0.5% in 88% of infertile and all of fertile cases whereas >0.5% in 12% of infertile cases during postovulatory phase. Chloride level estimation of cervical mucus is 96% accurate in infertile and 92% accurate in fertile cases compared to TVS. Keywords: Transvaginal Sonography, Sodium chloride, Ovulation, Cervical mucosa, Infertile, Fertile. INTRODUCTION Ovulation is a process resulting from hypothalamus, anterior pituitary and ovaries. The ovarian hormone influences the hypothalamo-pituitary axis to induce a FSH and LH surge, responsible for ovulation. Various procedures available for evaluation of ovarian function are vaginal cytology, basal body temperature recordings, premenstrual endometrial biopsy, steroid excretion estimation, LH surge test and ultrasonography for evidence of follicular rupture at time of ovulation [1]. Cyclic variations in the physiochemical properties of cervical mucus have been found from last few years. Various mucus studies are used in the diagnosis of various gynaecological disorders. At the time of ovulation, cervical mucus becomes copious, clear and watery. Peak of this mucus and peak of estrogen secretion occurs at same time [2]. Cervical mucus is a more sensitive indicator of estrogen activity [3]. Predictive method used for natural family planning is Billing's ovulation method in which development of changing mucus pattern has been considered [4]. Appearance of mucus has been correlated with the increasing estrogen level generated by the developing follicle. Mucus causes maximum wetness when luteinizing hormone is at maximum level. It has been considered that ovulation occurs an hour before to 48 hours after the peak symptom. Therefore, all the mucus wet days including peak symptom plus 72 hours following it are considered as fertile days of woman. Reason for infertility due to ovulatory failure has been found in 15-25% of infertile female population [5]. Rajani Shrivastava et al., Sch. J. App. Med. Sci., March 2016; 4(3C):781-785 782 Sodium Chloride (NaCl) is the major electrolyte component among the various constituents of cervical mucus [6]. At peak concentration of cervical mucus, NaCl can be responsible for positive fern test at the time of ovulation. NaCl coincides with the peak of fern like crystallization of mucus on glass slide [7]. Crystallization requires presence of mucin like substances as well as NaCl in high concentration [8]. On the basis of above considerations, study has been made to evaluate variations in chloride levels in preovulatory, ovulatory and premenstrual phase of the menstrual cycle and its utility in prediction and detection of ovulation. MATERIALS AND METHODS Study was conducted upon 100 women attending the outpatient department of Obstetrics and Gynaecology, G.R. Medical College & Kamla Raja Hospital, Gwalior, from September 2004 to March 2005. Women of reproductive age group with no obvious gynaecological or obstetric disease were included in the study. Detailed obstetric and gynaecological history of each patient was recorded with particular emphasis on menstrual cycles and period of infertility. Patients were investigated for Hb%, blood sugar, urine routine and microscopy, semen analysis, Kahn's test and venereal disease research laboratory test. Only women with healthy looking cervices and vagina were selected. Women with cervicitis, vaginitis, cervical erosion, previous electrocauterization of cervix and other such conditions were excluded from the study. Women were divided into two groups. Group I: Consisting of 50 infertile women in age group of 18 to 35 years, having regular menses with no associated clinically detectable abnormality. Group II: Consisting of 50 women of 18 to 35 years, who had two or more conceptions and were without clinically detectable disease. Patients were instructed not to have coitus 24 hours prior to the performance of mucus test, nor to use any kind of lubricant or douching before the study of cervical mucus since semen, prostatic secretions and other chemicals are known to interfere with the study of chloride levels. After recording the data of last menstrual period, each patient was called thrice. On each visit cervical mucus was studied for chloride concentration by test sheet method [9]. For collection of cervical mucus tuberculin syringe was used [10]. Test sheets were prepared by serially impregnating filter paper sheets with solutions of silver nitrate and potassium chromate. First visit was in immediate post menstrual period i.e. 2-3 days after stoppage of bleeding - on 6thto 9 th day of cycle. Second visit was in ovulatory period i.e. 13thto 16th day of cycle and third in premenstrual phase. i.e. 20th to 25th day of cycle. During these visits, transvaginal sonography was done which included follicular monitoring & endometrial pattern study in second visit and assessment of ovulation (follicular size and presence of free fluid in pouch of Douglas) in third visit. RESULTS Out of total 100women, 50 cases were infertile patients and 50 cases were fertile controls. Distribution of cases and controls according to chloride levels in preovulatory phase between 6thto 9thday of cycle was found as per Table 1. Distribution of cases and controls according to chloride levels in periovulatory phase between 13th to 16th days of cycle was found as per Table 2. Distribution of cases and controls according to chloride levels in postovulatory phase between 20th to 25th days of cycle was found as per Table 3. Distribution of cases according to follicular size as shown by TVS during ovulatory phase was found in following Figure 1. Distribution of cases according to endometrial pattern as shown by TVS during ovulatory phase and post ovulatory phase were found as per Table 4 and Table 5. Accuracy of chloride estimation for detection of ovulation in infertile cases and fertile cases were found as per following Figure 2. Table 1: Distribution of cases and controls according to chloride levels in preovulatory phase (between 6thto 9thday of cycle) Sr. No. Cases (Infertile cases) Control (Fertile cases) No. of cases % of cases Chloride level in % No. of cases % of cases Chloride level in % 1 10 20 0.1 10 20 0.1 2 38 76 0.3 38 76 0.3 3 2 4 0.5 2 4 0.5 Total 50 100 50 100 Rajani Shrivastava et al., Sch. J. App. Med. Sci., March 2016; 4(3C):781-785 783 Table 2: Distribution of cases according to chloride levels in periovulatory phase (between 13thto 16thday of cycle) Sr. No. Cases (Infertile cases) Control (Fertile cases) No. of cases % of cases Chloride level in % No. of cases % of cases Chloride level in % 1 1 2 0.1 - - 0.1 2 13 26 0.3 - - 0.3 3 13 26 0.5 20 40 0.5 4 20 40 0.7 20 40 0.7 5 3 6 0.9 10 20 0.9 Total 50 100 50 100 Table 3: Distribution of cases and controls according to chloride levels in postovulatory phase (between 20thto 25thday of cycle) Sr. No. Cases (Infertile cases) Control (Fertile cases) No. of cases % of cases Chloride level in % No. of cases % of cases Chloride level in % 1 6 12 0.1 10 20 0.1 2 38 76 0.3 40 80 0.3 3 6 12 0.5 - - 0.5 Total 50 100 50 100 Fig 1: Distribution of cases according to follicular size as shown by TVS during ovulatory phase Table 4: Distribution of cases according to endometrial pattern as shown by TVS during ovulatory phase Sr. No. Endometrial pattern Cases (Infertile cases) Control (Fertile cases) No. of cases % age No. of control % age 1. Proliferative Endometrium 36 72 46 92 2. Thin/proliferative/endometrium not corresponding to the phase of cycle 14 28 4 8 Total 50 100 50 100 Table 5: Distribution of cases and control according to endometrial pattern as shown by TVS during post ovulatory phase Sr. No. Endometrial pattern Cases (Infertile cases) Control (Fertile cases) No. of cases %age No. of cases %age 1 Secretory endometrium 34 68 46 92 2 Proliferative endometrium 12 24 4 8 3 Endometrial hyperplasia 4 8 - - Total 50 100 50 100 Follicular size> 16 mm 1 mm > more follicle each < 10 mm with 36 14 46 4 Distribution of cases according to follicular size as shown by TVS during ovulatory phase No. of cases No. of control Rajani Shrivastava et al., Sch. J. App. Med. Sci., March 2016; 4(3C):781-785 784 Fig 2: Accuracy of chloride estimation for detection of ovulation in infertile and fertile cases DISCUSSION Chloride levels in ovulatory cycle were found to be <0.5% in 96% of cases between 6th -9 th day of cycle, >=0.5% in 72% infertile and all of fertile cases between 13th -16th day of cycle, <0.5% in 88% of infertile and all of fertile cases whereas >0.5% in 12% of infertile cases between 20th -25th day of cycle. This difference in chloride levels from preovulatory, periovulatory to postovulatory phase was found to be statistically significant. These observations were in correlation with those of McSweeney et al.; who found change in spot intensity with a drop below 0.5% in the chloride concentration in post ovulatory phase of the cycle [9]. Hardy et al.; found that positive chloride spot disappear in post ovulatory phase of the cycle [11]. Also, Singh and Boss and Mathur et al.; found fall in chloride levels in post-ovulatory phase of ovulatory cycles [12, 13]. According to endometrial pattern, in ovulation phase, 72% infertile cases showed proliferative endometrium in concordance with follicular size of > 16 mm which is about to rupture whereas 92% of control cases showed the same. 28% infertile cases showed thin endometrium or endometrium out of phase having no dominant follicle preclusive of anovulatory cycles and only 8% showed the same in fertile group. In post ovulation phase, 68% infertile cases showed secretory endometrium which was taken as confirmative of ovulation and 92% in fertile cases showed the same. According to these results, 68% infertile cases had ovulatory cycle while 24% infertile cases showed proliferative phase whereas only 8% fertile cases showed proliferative endometrium which showed endometrial hyperplasia. Similarly, it was found by Parson et al. that 7-8% of women are anovulatory and anovulatory cycles were seen 3-6 times often in infertile women [14]. Moreover, as per Strowitzki T et al.; endometrium is definitely a fertility-determining factor [15]. Ovulation was detected in 72% of infertile and all of fertile cases by estimating chloride levels of cervical mucus. Taking ovulation &pre menstrual sonography as the standard parameter for detection of ovulation, ovulation was confirmed in 68% of infertile and 92% of fertile cases. Results of chloride level estimation of cervical mucus were thus found to be 96% accurate in infertile and 92% accurate in fertile cases. Fallacy rate was statistically insignificant (P=<0.5).However, it was noted that chloride level estimation was false positive in only 4% cases. Thus it is possible to locate the approximate day of ovulation by cervical mucus study and cases of sterility can be advised coitus on the appropriate days. Also the time for artificial insemination can be detected easily. Similar types of result were also found by Guida M et al. that urinary LH level determination yielded a 100% correlation with the simultaneous ultrasonographic diagnosis of ovulation. Mucus secretions and characteristics yielded a 48.3% correlation when simultaneously evaluated with ovulation. Betaglucuronidase levels yielded a 27.7% correlation. The salivary ferning test had a 36.8% ovulation-detection rate at the day of ovulation, but 58.7% of results were uninterpretable. Body temperature measurements yielded a 30.4% correlation with the simultaneous ultrasonographic diagnosis of ovulation [16]. CONCLUSION It has been concluded that chloride level estimation in the cervical mucus to detect ovulation offers a reliable and sought method in the treatment of infertility in developing country like India. Testing of the cervical mucus for chloride by “Spot Test Paper” can be used as means of appraising ovarian hormone activity which is simple, fairly reliable, easy to interpret, more economical, more rapid and can be done readily by the patient herself or in O.P.D.