Menstrual cycle-associated effects on some acute phase reactants parameters of Students in Usmanu Danfodiyo University Sokoto,North Western Nigeria

1. 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- α from macrophages and monocytes at the site of inflammatory lesions or infections [20].

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 ± 1.49 mg/L) was observed compared to luteal phase (1.54 ± 1.30 mg/L) in regularly menstruating women ( p = 0.0004). Similar observations were reported that CRP concentrations changed significantly during the menstrual cycle. The concentrations were highest in the early follicular phase and inversely correlated to estradiol concentration [22]. In regularly menstruating women CRP levels varied significantly across the cycle [23]. CRP tends to be lowest on the expected day of ovulation, and increased in the luteal phase. CRP was significantly positively associated with luteal progesterone. Moreover, a 10-fold increase in luteal progesterone was associated with a 19.4% increase in CRP. In another study among eighteen healthy regularly menstruating premenopausal women it was found that there was a 31% increase in the luteal phase in comparison to follicular phase levels [24]. Others studies discovered small non-significant differences in CRP concentrations between the follicular, ovulatory and luteal phase of the menstrual cycle in 36 healthy, young, normo-androgenic women, having a normal body mass index [25]. Similarly, a previous report [26] also observed a non-significant reduction in CRP levels from follicular to luteal phase of menstrual cycle. The differences between the results can most likely be explained by the differences in the assay methodologies used for the determination of CRP.

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 = faster/higher RBC sedimentation = higher ESR.) The sedimentation rate blood test measures how quickly red blood cells settle in a test tube in one hour. When inflammation is present in the body, certain proteins cause red blood cells to stick together and fall more quickly than normal to the bottom of the tube. The more red cells that fall to the bottom of a special test tube in one hour, the higher the sedimentation rate. These proteins (C-reactive protein) are produced by the liver and the immune system under many abnormal conditions, such as an infection, an autoimmune disease, or cancer. Moderately elevated ESR occurs with inflammation, but also with anaemia, infection, pregnancy, and old age. Females tend to have higher ESR and menstruation and pregnancy can cause temporary elevations [32]. The erythrocyte sedimentation rate, an indirect APR, reflects plasma viscosity and the presence of acute phase proteins, especially fibrinogen as well as other influences some of which are yet to be unidentified [33]. Some studies show significant difference in haematocrit and Erythrocyte Sedimentation Rate in the menstrual cycle [34].

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.

2. Materials and method

This study was carried out in Wammako Local Government situated within Sokoto metropolis. Sokoto State is located between longitude 11’30 ∘ , 13’50 ∘ East and latitude 4’to 6’0 ∘ North. It is bordered to the North by Niger Republic, Zamfara State to the East while Kebbi State borders most of the South and Western parts [35]. The State falls within the Savannah vegetation zone. Rainfall starts late and ends early with mean annual rainfall ranging from 500 to 1,300 mm, and annual average temperature of 28.3 ∘ C (82.9 ∘ F). It has a land area of about 28,232.37sq kilometres and stands at altitudes of 272 m above the sea level. The State has three peculiar seasons; the cold dry (December-February), hot dry (October-April) and wet. The wet season begins in most part of the State in May and lasts up to September. The vast Fadama land of the Sokoto Rima River system dissects the plain and provides rich alluvial soil and extensive grassland fit for a variety of crop cultivation, hence farming and livestock rearing are the principal activities in the State. The Metropolis was estimated to have a population of 427,760 people made up of Hausa and Fulani majority and a minority of Zabarmawa and Tuareg and other non-indigenous settlers. Based on 2015 population census [36]. Sokoto State had a population of 552,400. This was 0.303% of total Nigeria population with an average estimate of 564,585 in 2017 based on the population annual growth rate of 1.1%.

3. 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).

4. 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.

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 ( p = 0.00) and WBC count ( p = 0.03) in both Follicular and Luteal Phases of Menstruation.

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 ( $p>$ 0.05).

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 ( $p>$ 0.05). However, 93.3% of the study participants were single while 6.7% were married.

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 ( p = 0.00 and 0.03 respectively). The difference in ESR may be due to increase in rouleaux formation in the follicular phase due to decrease in the red blood cells and increase in plasma ratio during the early follicular phase (menstrual phase). Our finding agrees with the findings in a previous study in Benin [34]. The difference in total leucocyte count may due to tremendous increase in granulocytes during menstruation which agrees with the finding in a study in Benin [40]. However, there was no statistically significant difference in the serum albumin, C-reactive protein, and the remaining full blood count parameters when the follicular and luteal phase of menstruation was compared. Our finding agrees with a previous report in India [41]. Our finding is however at variance with findings in previous reports [30, 34].

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 ( $p>$ 0.05). In both phases there were no clinically significant differences between the parameters. Although there are no published articles to substantiate the finding in this study.

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.

6. 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.