Fluctuation of Serum Leptin Through Menstrual Cycle in Young Fertile Women in Mosul City

Background: Leptin is a hormone secreted by adipocytes , it is aprotein encoded by the ob gene. Mutations in the ob gene in animals cause obesity , leptin affects nutritional homeostasis and reproductive function. Objectives : a followup study was done to investigate serum leptin levels in 30 fertile healthy normal weight women with regular menstrual cycle aged (8-45) years in folicular and luteal phase of menstrual cycle. Methods: Fasting blood samples were obtained from normal weight fertile women(BMI 19.5-24.9 Kg/m 2 ) to measurement serum concentrations of leptin, which measured by using( ELISA) in follicular phase (2 nd day of menstriual cycle ) then ask the women to come back and give another blood sample in 21 st 23 nd day of the cycle to measure serum progesterone and leptin in luteal phase of the menstrual cycle. Results: The mean age ± SD of the normal fertile women was (29.1± 8.4) years, the mean weight value ±SD was (53±5)kg and the mean height value±SD was(158 ± 6.2) cm and the mean BMI±SD was (21.2± 1.3). Leptin was higher in luteal phase than follicular phase in normal weight fertile women ,this was statistically significant (p≤ 0.000), the mean ± SD of progesterone hormone was (7.8 ± 3.2 ) in day 21-23 of the cycle this was the indicator for ovulation and starting of luteal phase. Conclusion: A great variation in serum leptin levels throughout the menstrual cycles with lower levels in the follicular rather than luteal phase in normal weight fertile women is found.


INTRODUCTION
t Jackson Laboratory in 1950 the effects of leptin were observed by studying mutant obese mice that arose at random within a mouse colony 1 .These mice were very obese and extremely voracious. , many strains of laboratory mice have been found to be homozygous for single-gene mutations that cause them to become grossly obese, and they fall into two classes:"ob\ob", those having mutations in the gene for the protein hormone leptin, and "db\ob ", those having mutations in the gene that encodes the receptor for leptin , When ob\ob mice are treated with injections of leptin, they lose their extra fat and return to normal body weight. Leptin itself was discovered in 1994 by Jeffrey M.Friedman and colleagues at the Rockefeller University through the study of such mice 2,3 .
Leptin is a 16 KDa protein produced by adipose tissue and secreted into the peripheral blood 2,3,4 .
It is apparently paradox that leptin which was named after the Greek word (Leptos )for lean is encoded by the obesity gene (ob).This unfortunate misnaming occurred because it is leptin deficiency that was discovered first in obese mice 2,5,6 .
Leptin interface with the reproductive axis at multiple sites, it has stimulatory effects at the hypothalamus and pituitary and inhibitory actions at the gonads 7,8 .
In women the reproductive system , not like that of men, has regular cyclic changes which may be regarded as periodic preparations for fertilization and pregnancy. In humans and other primates, the cycle is a menstrual cycle, and its most distinct feature is the periodic vaginal bleeding that occurs with the take of the uterine mucosa (menstruation) 9 .
In advanced researches , leptin has been found to play a role in other target reproductive organs such as the endometrium, placenta and mammary gland; with corresponding influences on important physiologic processes such as menstruation, pregnancy and lactation 7,9 . It may act as the important connection between adipose tissue and the reproductive system, suggesting there are adequate energy reserves are present for good and normal reproductive function, suboptimal nutritional status as functional hypothalamic amenorrhea, exercise-induced amenorrhea and eating disorders are associated with low serum leptin levels and conditions with extra energy stores or metabolic disturbances such as obesity and polycystic ovarian syndrome which usualy have high serum leptin levels, making the possibility that moderate leptin deficiency or resistance may be at least responsible for the reproductive abnormalities that occur with these conditions 7,10 .

Subjects & Study Design
The study was conducted at the women health center of al-khansa hospital in 2013 a follow up prospective study included thirty fertile apparently healthy women in reproductive age (18 -45) years old.
All women included in the study were interviewed and the general information was taken to fill the questionnaire .
A verbal informed consents were taken from all participants They had regular menstrual cycle for the last 6 months ago , and did not used any hormonal medication for at least 2 months before the study. None of them had a history of coronary heart disease, diabetes mellitus, Hypertension, they were neither pregnant nor lactating.
For every women height was measured to the nearest millimeter using a standard stadiometer, with the participant bare foot and wearing light weight clothing, weight was measured to the nearest (0.1)kg using a standard digital scale and BMI (kg/m 2 ) was computed by dividing the weight in kilogram (kg) over the height in square meters (m 2 ).

SPECIMENS
The subjects were instructed to have an overnight fasting, through antecubital venepucture blood samples were obtained from all women participated in the study. Two milliliter (2ml) of venous blood had been collected in plain tube in 2 nd -3 rd day of menstrual cycle then incubated for 37C for 15 minutes in water bath, after centrifugation for 10 minutes at 3000 rotation per minute (rpm), aspiration of supernatant sera were put in plane tube , then freezed at -20C till time of assay to measure leptin hormone .
Then the women were instructed to come back and give another blood sample in 21 st -23 nd day of the cycle to measure serum progesterone and leptin in luteal phase of the menstrual cycle. .

Leptin Measurement
Leptin was measured by using kit provided by DRG company, (Germany) Based on a solid phase ELISA of a sandwich type. The analytical sensitivity of zero standard was equal to (1.0ng/mL) and the assay dynamic range was(1-100ng/mL).

Progesterone Measurement
VIDAS progesterone kit provided by Biomerieux (France) is automated quantitative test for measurement of progesterone in human serum or plasma using the ELFA technique (Enzyme linked fluorescent Assay) .

Statistical Analysis
SPSS version 11.5 was used for the statistical analysis. 1. Descriptive results were represented as mean ± standard deviation (SD). 2. The comparisons within the group were done by student t-test .

RESULTS
1. The anthropometric parameters of 30 normal weight fertile women is shown on Table1 .  Table 2 .

Leptin in follicular phase and luteal phase of menstrual cycle in normal fertile women
Serum leptin was higher (p≤ 0.000) in luteal phase than follicular phase in normal weight women, the mean ± SD of serum progesterone hormone was (7.8±3.2 ) ng/ml in day 21-23 of the cycle this was the indication for ovulation and luteal phase starting table 2 .

Table 2
Leptin in normal weight with regular menstrual cycle women in the follicular phase and luteal phase.

Parameters
Normal weight with regular cycle women (n = 30)

*p -value
Mean± SD at 2nd day of cycle (follicular phase) Mean± SD in (luteal phase)

DISCUSSION
Leptin adipocyte-derived hormone is one of these cues which have been studied carefully in the regulating the reproductive physiology. Humans and mice lacking leptin or leptin receptor are infertile. Administration of Leptin to leptin-deficient subjects and ob/ob mice induces puberty and restores fertility 11 .
The role of leptin in the preservation of body weight, related with various other regulatory functions in endocrine systems including the hypothalamo-pitutary gonadal axis which is well recorded. New works reveal that leptin is also involved in reproduction and responsible for fertility of humans and animals 12 .
Leptin level enlightin the brain that metabolic reserve are adequate for reproduction 13 .
It appears obvious that a minimum threshold of serum leptin is essential for a regular ovulatory function 14 . The mechanism of high serum leptin in luteal phase is not known exactly . It could be by increased a adipocyte production of leptin hormon due to high caloric intake or hypothalamic neuropeptide Y production. Alternatively, this increase may indicate the release of leptin from mature ovarian follicles 15 .
Low serum leptin is well known in amenorrhea which found in conditions such as in highly trained women or in women with anorexia nervosa. Thus, leptin clearly appears to be linked to the reproductive system, and menstrual cycle 14 .
In the present study, a thirty fertile women with normal bodyweight and regular cycles, in whome luteal phase confirmed by serum progesterone estimation, luteinization and ovulation were considered to have taken place when serum progesterone concentration exceeded 5 ng\ml 16,17 .
The mean ± SD of progesterone level was (7.8 ± 3.2) ng\ ml in the (21 -23) day of the cycle .
The serum leptin level in the 2 nd day of the cycle (follicular phase) was lower than it in the 21-23days (luteal phase )of the menstrual cycle , this difference was statistically significant (p< 0.0001) , so this study demonstrated for the first time in our locality that healthy women in mosul city with normal weight having significantly higher serum leptin level during luteal in contrast to the follicular phase. This result is in agreement with a number of other studies 15,17,18,19,20,21,22,23,24 had shown considerable variation in serum leptin levels throughout the menstrual cycles with higher levels in the luteal rather than follicular phase . Einollahi  Leptin homone act as mediator of cell proliferation and with respect to the presence of leptin receptors in the endometrium, its rational that the luteal phase of menstrual cycle has a high leptin levels, also preovulatory surge of estrogen might increase the circulating leptin which in turn could provoke the gonads to produce more leptin 25,26,27 .
Data of the present study seem to contradict the finding of other studies, which found no statiatical significant difference in serum leptin level in follicular and luteal phase, Shafi and Afzal in 2009 study 83 women in reproductive age years, 37 were in follicular phase ,47 in luteal phase no significant difference found in leptin level (54.6 ±33.4 ; 53.5± 38.8) respectively (p≤ 0.25 ) but when they classified them according to BMI they found no significant difference in overweight women and only normal weight women show higher serum leptin in follicular phase 28 .
Some other studies also have found either small but insignificant swing toward higher leptin levels at the end of the cycle 29 . Serum leptin concentrations were the same at the beginning of the cycle, around the time of the anticipated ovulation and at the end of the cycle (no fluctuation at all) 30,31 .
Recommendations and Further Work 1. To study leptin hormone in fertile and non fertile women.

2.
To study leptin hormone in pregnant women and after delivary.