Abstract
BACKGROUND:
The menstrual cycle is the cycle of natural variations that occurs in the uterus and ovary as an essential part of making sexual reproduction possible. It is characterized by hormonal changes but the changes that occur in some active phase reactants (APR) parameters have not been fully elucidated.
OBJECTIVE:
The aim of this study was to compare the serum albumin, ESR, and C-reactive protein levels in follicular and luteal phases of menstruation.
METHODS:
A total of 90 healthy regularly menstruating women where used for this study. Forty-five of the study participants were in their follicular phase while the other 45 where in their luteal phase. Four milliliters of blood were withdrawn from each patient under aseptic condition and two milliliters was dispensed into EDTA container while the other two milliliters were dispensed into a plane container. The EDTA anticoagulated blood was used for ESR and full blood count while the serum from the plain tubes was used for analysis of C-reactive protein and Serum Albumin. Sysmex K-3 auto-analyser (Sysmex, Kobe Japan) was used for te determination of full blood count, the Westergren method was used for ESR estimation, Bromo Cresyl Green method was used for serum albumin and ELISA method was used for CRP determination. Data analysis was carried out using SPSS version 23.
RESULTS:
This study showed a statistically significant difference in the ESR (
CONCLUSIONS:
There is need to generate data on menstrual disorders and their impact on women’s health status, quality of life and social integration. It is vital that evaluation and treatment of menstrual complaints should be given a higher priority in primary care programs. There is need to invest in public enlightenment program to increase awareness in secondary schools to increase the level of awareness among adolescents as well as young females.
Introduction
Menstruation is the periodic change occurring in females, which results in the shedding of blood and endometrium from the uterine cavity, and which may be associated with various constitutional disturbances. Premenstrual syndrome (PMS) is a collection of physical and psychological symptoms that some women experience during the late luteal phase of each menstrual cycle (7 to 14 days prior to menstruation).
Menstruation also known as period, monthly flow or menses is the regular discharge of blood and mucosal tissue (known as menses) from the inner lining of the uterus through the vagina [1]. The first period usually begins between twelve and fifteen years of age, a point in time known as menarche [2]. Bleeding usually lasts around 2 to 7 days [1]. Menstruation stops occurring after menopause, which usually occurs between 45 and 55 years of age [3]. Menstrual periods may occasionally start as young as eight years old and still be considered normal [1]. The typical length of time between the first day of one period and the first day of the next is 21 to 45 days in young women, and 21 to 31 days in adults (an average of 28 days) [1, 4]. Periods also stop during pregnancy and typically do not resume during the initial months of breastfeeding [1]. Up to 80% of women report having some symptoms prior to menstruation [3]. The menstrual cycle is commonly divided into three phases: the follicular phase, ovulation, and the luteal phase [5]. This Follicular phase is also called the proliferative phase because a hormone causes the lining of the uterus to grow, or proliferate, during this time [6]. Through the influence of a rise in follicle stimulating hormone (FSH) during the first days of the cycle, a few ovarian follicles are stimulated [6]. As they mature, the follicles secrete increasing amounts of estradiol, an estrogen. The estrogens initiate the formation of a new layer of endometrium in the uterus, histologically identified as the proliferative endometrium. The estrogen also stimulates crypts in the cervix to produce fertile cervical mucus, which may be noticed by women practicing fertility awareness [7].
The ovulation phase occurs in the middle of the cycle and at this point a mature egg will ovulate (be released) from a follicle. This usually occurs around cycle day 14 in a normal 28-day menstrual cycle [5]. The Luteal phase is the second half of the cycle and it’s during this phase that an embryo may or may not implant onto the uterine wall. If embryo implantation occurs, a pregnancy will begin. If an embryo doesn’t implant, hormone levels will drop and a new cycle will begin [5]. The menstrual cycle is the cycle of natural variations that occurs in the uterus and ovary as an essential part of making sexual reproduction possible. Each cycle can be divided into three phases based on events in the ovary (ovarian cycle) or in the uterus (uterine cycle) [8]. The ovarian cycle consists of the follicular phase, ovulation, and luteal phase whereas the uterine cycle is divided into menstruation, proliferative phase, and secretory phase. Both cycles are controlled by the endocrine system and the normal hormonal variations that occur can be interfered with using hormonal contraception to prevent reproduction [9].
During the follicular phase, estradiol suppresses the production of luteinizing hormone (LH) from the anterior pituitary gland. When the egg has nearly matured, levels of estradiol reach a threshold above which they stimulate production of LH. These opposite responses of LH to estradiol may be enabled by the presence of two different estrogen receptors in the hypothalamus: estrogen receptor alpha, which is responsible for the negative feedback estradiol-LH loop, and estrogen receptor beta, which is responsible for the positive estradiol-LH relationship [10]. In the average cycle this LH surge starts around cycle day 12 and may last 48 hours. The release of LH matures the egg and weakens the wall of the follicle in the ovary, causing the fully developed follicle to release its secondary oocyte [6]. The secondary oocyte promptly matures into an ootid and then becomes a mature ovum. The mature ovum has a diameter of about 0.2 mm [11]. Which of the two ovaries – left or right – ovulates appears essentially random; no known left and right co-ordination exists [12]. Occasionally, both ovaries will release an egg [12], if both eggs are fertilized, the result is fraternal twins [11].
The luteal phase is also called the secretory phase. An important role is played by the corpus luteum, the solid body formed in an ovary after the egg has been released from the ovary into the fallopian tube. This body continues to grow for some time after ovulation and produces significant amounts of hormones, particularly progesterone [6]. Progesterone plays a vital role in making the endometrium receptive to implantation of the blastocyst and supportive of the early pregnancy; it also has the side effect of raising the woman’s basal body temperature [7]. There is a noted secretion of prolactin towards the end of the secretory phase.
Acute-phase proteins are large and varied group of glycoproteins in serum released into blood stream in response to a variety of stressors. All the up-regulated proteins have been called positive APP, in order to differentiate them from the so-called negative APP that is down-regulated [13]. An acute-phase protein concentration may either increase (positive acute-phase proteins) or decrease (negative acute-phase proteins) by 25 percent during inflammatory disorders [14]. Negative acute phase reactants (albumin, transferrin, transerythrin, retinol-binding protein, antithrombin, transcortin) decreases during inflammation [15]. The decrease of such proteins may be used as markers of inflammation [16].
Acute Phase Reactants refers to protein components of plasma whose concentration is significantly increased in acute phase of inflammatory processes and tissue injury [17].
Positive APP is represented by large number of proteins which may be divided into three classes based on their normal plasma concentrations. Concentration of some proteins like C reactive protein (CRP), Serum amyloid A (SAA) rise as early as 4 hours after inflammatory stimulus and attain their maximum levels within 24 to 72 hours and also decline very rapidly. Class II APP begin to increase 24 to 48 hours and reach to their maximum level in about 7 to 10 days, about two weeks are required to return to their normal levels [18]. Elevated expressions of APP differ widely from species to species and their pattern often depends upon gender.
Positive APPs are C-reactive protein (CRP), D-dimer protein, mannose-binding protein (MBP), alpha 1 antitrypsin, alpha 1 antichymotrypsin, alpha 2 macroglobulin, fibrinogen, prothrombin, factor VIII, von-Willebrand factor, plasminogen, complement factors, ferritin, serum amyloid P component (SAP) complement, serum amyloid A (SAA), ceruloplasmin (Cp), and haptoglobin (Hp) [19]. APR is stimulated by the release of cytokines such as IL-1, IL-6, and TNF-
Various researches have reliably demonstrated the impact of CRP levels [21]. However, the researches related to phases of menstrual cycle and its effect on serum CRP has been scarce and inconsistent. CRP is normally present in very small amount in serum and its levels rises quickly and significantly in various infectious and inflammatory conditions. It is an indicator for low level of persistent inflammation [21]. A significant raised mean CRP levels in follicular (1.88
Human Serum Albumin is a single-chain, non-glycosylated polypeptide with a molecular weight of 66,500 Da containing 585 amino Acids [27]. Serum albumin level accounts for about 50–60% of the serum proteins with a range of 35–50 g/L [28]. Protein depletion results in hypoproteinemia with many pathologic changes including degeneration of the connective tissue of the gingival and periodontal ligament, osteoporosis of the alveolar bone, impaired deposition of the cementum, delayed wound healing, and atrophy of the tongue epithelium. It has also been demonstrated that older adults with vertical tooth mobility and periodontal pockets greater than 6 mm had a significantly lower albumin concentration. Studies have also described the negative association between albumin concentration and periodontal diseases in their respective studies [29]. The Serum albumin concentrations are sometimes used to indicate the degree of haemodilution [30]. In some studies albumin concentration was significantly increased in the luteal compared to the follicular phase [31].
Erythrocyte sedimentation rate (ESR) is the measure of red blood cells that settle in a test tube over a set time period. It is a very vague measure of systemic inflammation. (Greater inflammation
There is paucity of data on acute phase reactants in follicular and luteal phases of menstruation among women and girls in Nigeria. The natural process of menstruation comes as a big challenge to women and girls in many communities in Africa putting an addition burden on women. It is not known if a relationship exists between inflammatory markers (CRP, Albumin and ESR) and menstrual cycle. The aim of this study was to compare the serum albumin, ESR, and C-reactive protein levels in follicular and luteal phases of menstruation among female Students in Usmanu Danfodiyo University Sokoto, North Western Nigeria.
Materials and method
This study was carried out in Wammako Local Government situated within Sokoto metropolis. Sokoto State is located between longitude 11’30
Study design
This case study involved ninety (90) consecutively selected healthy regularly menstruating female subjects to assess the level of some selected acute phase proteins, some full blood count parameters and ESR in follicular and luteal phases respectively. Blood obtained was tested for Serum albumin, C-reactive protein, full blood count and ESR. Results of these parameters were analyzed using Statistical Package for Social Sciences (SPSS) version 23.
Study population
The target populations for this study were regularly menstruating female students of Usmanu Danfodiyo University Sokoto who have attained the age of menarche (16–45) years. A total of 90 subjects were recruited for this study of which 45 were in their follicular phase while the other 45 were in their luteal phase.
Study subjects
Inclusion criteria
Females who had menstrual cycle length of 24–38 days with flow of 4–8 days and variation of 0–20 days from cycle to cycle were considered as regularly menstruating women. Consenting females aged between 16–49 years who were on their luteal and follicular phases attending Usmanu Danfodiyo University Sokoto, Nigeria was included in this study.
Exclusion criteria
Non-consenting, regularly menstruating female students outside luteal and follicular phases and those below 16 and above 49 years of age and those who have been on contraceptives in the last three months were excluded from this study.
Sample size determination
The sample size was calculated according to Co- chran [37] formula below:
Therefore
Therefore
0.05
The sample size was limited to ninety subjects. This was due to financial constraints and the limited time frame required for the completion of this study.
Sampling and techniques
Sample collection
Four milliliters (4 ml) of whole blood was collected via venipuncture using BD vacutainer system. Part of the blood was dispensed into K
Methods of analysis
The following techniques were used to measure full blood count parameters and erythrocyte sedimentation rate parameters, C-reactive protein and albumin. For ESR, the Westergreen technique was used to analyze the EDTA anticogulated blood. For serum albumin the bromo-cresyl green technique was used and for C-reactive protein an ELISA kit was used according to the manufacturer’s instruction. The Full Blood Count was determined using the three-part differential fully automated Sysmex Haematology Analyzer (Sysmex KX-21N Model, Kobe, Japan).
Erythrocyte Sedimentation Rate (Westergreen’s technique) [38]
Principle of test
When EDTA blood in a vertical positioned in a Westergreen’s pipette and left undisturbed, red cell aggregate, stack together to form a rouleaux, and sediment through the plasma. The ESR is the rate at which this sedimentation occurs in 1 hour as indicated by the length of the column of clear plasma above the red cell, measured in mm. High temperature (over 25
Serum Albumin (Bromocresol Green Method) [39]
Principle: Under acidic condition (between pH 3.5–4.2), serum albumin binds specifically with bromocresol green (BCG) to form a blue-green coloured complex which will be measured spectrophotometrically at 600 nm.
Determination of serum C-reactive protein using ELISA
Principle: The C-reactive protein ELISA kit is a solid phase direct sandwich method. The samples and anti CRP-HRP conjugate are added to the wells coated with Mab to CRP. CRP in patient’s serum binds to anti-CRP Mab on the well and anti-CRP second antibody then binds to CRP. Unbound protein and HRP conjugate are washed off by wash buffer. Upon the addition of substrate, the intensity of color is proportional to the concentration of CRP in the samples, A standard curve is prepared relating color intensity to the concentration of CRP.
Questionnaire
A semi structured interviewer-administered questionnaire was administered to all consenting participants to obtain information on the subjects’ bio-demographic data.
Informed consent
Written informed consent was obtained from all the participants.
Statistical analysis
Data was analyzed using SPSS Version 23 (SPSS Incorporated, Chicago, IL, USA). The result was expressed as mean
Results
A total number of 90 healthy regularly menstruating female study participants were recruited in this study. Of this number, 45 participants were in their follicular phase and 45 participants were females. The age range of these participants was 16–49 years.
Serum Albumin, ESR and C-reactive protein level in follicular phase and luteal phase
Serum Albumin, ESR and C-reactive protein level in follicular phase and luteal phase
Table 1 shows the effect of the menstrual cycle phases of the ESR, Albumin and CRP in the Follicular Phase and Luteal Phase. There was only a statistically significant difference in the ESR (
Table 2 shows the correlation between Serum Albumin, C-reactive protein, ESR and some full blood count Parameters in the Follicular phase of the menstrual cycle. There was no significant difference in changes when serum albumin, C-reactive protein and ESR was compared to some full blood count parameters in Follicular Phase.
Table 3 shows the correlation between Serum Albumin, C-reactive protein, ESR and some full blood count Parameters in the luteal phase of the menstrual cycle. There was no significant difference in changes when serum albumin, C-reactive protein, ESR was compared to some full blood count parameters in the Luteal Phase (
Table 4 shows the effect of socio-demographic data on Serum Albumin, C-reactive protein, ESR and some full blood count Parameters. There was no significant effect of age and marital status on the Serum Albumin, C-Reactive Protein, ESR, and full blood count parameters in the Follicular and Luteal phase (
Correlation between serum albumin, C-reactive protein, ESR levels and some full blood count parameters in follicular phase
Key: HCT – haematocrit, HGB – haemoglobin, RBC – red blood cell count, WBC – white blood cell count, PLT – platelet count, ESR – erythrocyte sedimentation rate,
Correlation between serum albumin, C-reactive protein and ESR levels in luteal phase
Key: HCT – haematocrit, HGB – haemoglobin, RBC – red blood cell count, WBC – white blood cell count, PLT – platelet count, ESR – erythrocyte sedimentation rate,
The effect of socio demographic data on serum albumin, C-reactive protein, ESR, and some selected full blood count parameters
The changes in concentrations of the female sex hormones during each phase of the menstrual cycle contribute to the changes in some biochemical parameters. The effect of menstruation and also some other factors such as female sex hormones (oestrogen, follicle stimulating Hormone, luteinizing hormone and progesterone) interplays and could possibly affect some biochemical parameters. Biochemical parameters are indicators of health and nutritional status of a woman and in turn affect her reproductive capability [34].
This research work focused on ninety (90) study participants who were either in the follicular or luteal phase of their menstrual cycle in Usmanu Danfodiyo University Sokoto. Distribution based on marital status indicated that 6.7% of the study participants were married while the remaining 93.3% were single. The participants were tested for full blood count, Serum Albumin, C-reactive protein and ESR.
Haematological analyses of the study participants in this study showed that there were statistically significant differences in the ESR and WBC in both phases (
In this study, there were no statistically significant as correlation between Serum albumin, C-reactive protein, ESR and full blood parameters in follicular and luteal phase (
In this study, there was no statistically significant difference in the full blood count parameters, Serum Albumin, C-reactive protein and ESR based on age and marital status of the study participants in the follicular and luteal phase. This may be due to decreased number of married study participants in the study.
Conclusion
This study has indicated that there was statistical difference in ESR and WBC count when the follicular phase and luteal phase were compared. There was no statistically significant difference in the acute phase proteins and full blood count parameters based on age and marital status among women in the follicular phase and luteal phase phases of their menstrual cycle.
Footnotes
Conflict of interest
The authors confirm that there is no conflict of interest associated with this paper.
