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 Table of Contents  
Year : 2021  |  Volume : 16  |  Issue : 1  |  Page : 80-85

The variation of neutrophil count at different phases of the menstrual cycle in periodontally healthy females

1 Department of Periodontology, Yogita Dental College and Hospital, Ratnagiri, Maharashtra, India
2 Department of Periodontology, SEGI University Kota Damansara, Petaling Jaya, Selangor, Malaysia
3 Currently a Private Practioner with Specialization in Periodontal Surgeries and Implantology

Date of Submission19-Mar-2019
Date of Decision20-Nov-2020
Date of Acceptance11-Jan-2021
Date of Web Publication29-Jul-2021

Correspondence Address:
Dr. Aishwarya Hajare
Department of Periodontology, Yogita Dental College and Hospital, Khed, Ratnagiri, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jdmimsu.jdmimsu_60_19

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Introduction: The effect of different levels of sex hormones in the body is to bring out changes in every part of the body. The changes occurring in periodontium under the influence of female sex hormones during menstrual cycle is important from the perspective of periodontal health. Aim: The purpose of this study is to compare the neutrophil count at different time points during menstrual cycle in GCF and to compare the neutrophil count with clinical parameters. Materials and Method: Fifty five periodontally healthy women with regular menstrual cycle were included in the study. Clinical parameters recorded were Gingival bleeding index (GBI) and the Modified Gingival index (MGI) at each phase of menstrual cycle(Ovulatory phase OP, Menstrual phase MH and Premenstruation phase PMP). Gingival crevicular fluid (GCF) was collected at each study phase for analysis ofneutrophil count. Results: Both the GBI and the MGI increased remarkably during the menstrual cycle, and were significantly higher during ovulation than in menstruation and premenstruation phase. The level of neutrophils increased during the different phases of menstrual cycle, highest count being observed during the ovulatory phase. Conclusion: It was concluded that periodontal health is altered during different phases of menstrual cycle.

Keywords: Gngival crevicular fluid, menstrual cycle, neutrophil count

How to cite this article:
Hajare A, Shetty P, Jadhav V, Yadav S, Pardeshi P, Patel S. The variation of neutrophil count at different phases of the menstrual cycle in periodontally healthy females. J Datta Meghe Inst Med Sci Univ 2021;16:80-5

How to cite this URL:
Hajare A, Shetty P, Jadhav V, Yadav S, Pardeshi P, Patel S. The variation of neutrophil count at different phases of the menstrual cycle in periodontally healthy females. J Datta Meghe Inst Med Sci Univ [serial online] 2021 [cited 2021 Sep 23];16:80-5. Available from: http://www.journaldmims.com/text.asp?2021/16/1/80/322642

  Introduction Top

Hormones are signaling molecules that are produced by endocrine glands of the body to regulate physiology and behavior of the cells. Their effect reflects physiological and pathological changes in almost all types of tissues of the body. The anterior pituitary gland secretes follicle-stimulating hormone (FSH) and luteinizing hormone (LH) into the bloodstream which stimulates the ovaries to secrete estrogen and progesterone.[1] The hormones such as FSH, LH, estrogen, and progesterone play an important role in different phases of the menstrual cycle, namely menstrual phase, follicular phase, ovulatory phase, and luteal phase.[1] Variations that occur in the levels of estrogen and progesterone during the menstrual cycle may influence other systems of the body as well.[2],[3],[4],[5],[6],[7],[8] The presence of receptors of estrogen and progesterone on the cells of gingiva, osteoblasts, and fibroblasts brings out changes in these tissues owing to the change in the concentration of these hormones.[2],[3]

Progesterone is secreted by the corpus luetum after ovulation on the 14th day of the menstrual cycle. It causes the dilation of small blood vessels, thereby increasing the vascular permeability.[5] The increase in vascular permeability affects the activity of polymorphonuclear (PMN) cell and alters the serum level of inflammatory cytokines. Progesterone also plays an important role in the production of prostaglandin E2.[9],[10]

Estrogen is secreted by follicles in the ovaries. It suppresses the production of leukocytes from the bone marrow and increases the polymorphonuclear phagocytosis.[11],[12],[13]

Both progesterone and estrogen may amend various immunologic responses including antigen expression and presentation, cytokine production as well as the expression of apoptotic factors leading to cell death.[14]

Thus, there are several types of gingival diseases in which alterations in levels of female sex hormones namely, estrogen, progesterone, FSH, and LH are considered to be either initiating or complicating factor in periodontal disease. This association is evident in periodontal disease classification which includes hormone-related disease categories: puberty-associated gingivitis and pregnancy-related gingivitis.[15]

The standard menstrual cycle consists of duration of 28 ± 3 days, which comprises follicular phase, ovulation phase, luteal phase, and menstrual phase. During these phases, the female sex hormones fluctuate wherein the first peak of estrogen is observed before the ovulation day and the second peak is observed in the luteal phase, whereas for progesterone, the increase is observed in the luteal phase.[16]

The incidence of gingivitis in puberty is elevated due to alterations in the levels of the female sex hormones that induces inflammatory changes and changes in the host response which are more severe than that observed in adults with the same amount of plaque.[17],[18]

Interestingly, the cells depicting the risk for developing periodontal disease are PMN lymphocytes in gingival crevicular fluid (GCF). These cells are known to play a central role in the pathogenesis of periodontitis and their number varies considerably from patient to patient and from site to site in the same mouth.[19]

Studies conducted to analyze the effect of the menstrual cycle on the periodontium showed variable results. Some of these studies indicated that there was an increase in GCF flow on the day of ovulation compared to the menstrual phase of the cycle.[20] Machtei et al. found that the gingival index was higher in the ovulation phase.[21],[22] Baser et al. and Buduneli and Kinane reported an increase in bleeding on probing (BOP) and increase in the levels of interleukin-1 β.[23],[24] On the contrary, Becerik et al. believed that sex-hormone fluctuations during the menstrual cycle exert no effect on the concentration of cytokines in GCF.[25] Khosravisamani et al. stated that levels of description interleukin-1 β and tumor necrosis factor-α increased during different phases of the menstrual cycle.[26]

In all stages of pregnancy, menopause and puberty do induce inflammatory changes in the body. Thus, the present study was conducted to evaluate neutrophil count in GCF at different phases of the menstrual cycle and correlate it with periodontal parameters using gingival bleeding index (GBI) and modified gingival index (MGI).

  Materials and Methods Top

Study design and eligibility criteria

The present study was conducted on 55 female students of Yogita Dental College and Hospital, Khed, Maharashtra.

Inclusion criteria

  • Females with age 18–25 years periodontally healthy subjects with no inflammatory lesions of the mouth
  • Females with the regular menstrual cycle of 28–30 days in their last three periods and duration of menstruation of 5–7 days
  • The GBI (Ainamo and Bay) showing < 10% of sites with BOP within 10 s after probing
  • Females who have not undergone scaling and root planing and/or periodontal tissue surgery at least 6 months before the start of the study.

Exclusion criteria

  • Females using any type of contraceptive drugs and/or medications that affect the sex hormones
  • Use of antibiotics and nonsteroidal anti-inflammatory drugs 3 months before the start of the study
  • Pregnant or breastfeeding females
  • Females with any history of systemic diseases
  • Females with severe dental crowding or using any orthodontic appliance or any dental prosthesis
  • Females using tobacco in any form.

Study protocol

The study was ethically approved by the Ethical Committee of Yogita Dental College and Hospital, Khed. The CONSORT guidelines were followed in the study protocol. Written consent was signed by all the participants before the commencement of the study. The females who fulfilled the inclusion criteria were enrolled in the study. The periodontal parameters were evaluated, and if BOP was found in <10% of sites probed (BOP <10%), the participant was considered as periodontally healthy and included in the study.

Clinical parameters

Clinical parameters such as GBI and MGI were recorded at each phase of the menstrual cycle, i.e., ovulatory phase (OP), menstrual phase (MH), and premenstruation phase (PMP). The GBI by Ainamo and Bay in 1976 was recorded by gentle probing of the orifice of the gingival crevice. If bleeding occurred within 10 s, a positive finding was recorded and the number of positive sites were recorded and then expressed as a percentage of the number of sites examined.[27] The MGI devised by Lobene et al. in 1986 was recorded to assess the gingival inflammation by examining the gingiva and then assigning respective scores for the gingival inflammation.[28]

Gingival crevicular fluid collection

The GCF was collected in the morning, 2–3 h after breakfast. The gingiva was air-dried and isolated with cotton pellets for 1 min prior to sampling. The supragingival calculus if present was removed with a sterile curette. A 7 mm × 2 mm dimension of Durapore filter paper with a pore size of 0.22 μm (hydrophilic membrane filters of polyvinylidene difluoride, Merck Life Science Private Limited, India) was inserted into the gingival sulcus until mild resistance was felt and was kept for 10 s in the sulcus. The GCF sample was collected from the mesiobuccal sulcus of first and second maxillary molars. Sites which did not express GCF or sites contaminated with blood and saliva were excluded.

Gingival crevicular fluid neutrophil determination

The filter paper containing GCF was immediately suspended into sealable plastic siliconized polypropylene tubes containing a solution of 40 μl phosphate-buffered saline without calcium, and 3 mm ethylenediaminetetraacetic acid was mixed with 1% bovine serum albumin. The tubes were vortexed for 30 s, and then, paper strips were removed from the solution. Twenty microliter of the cell suspension was then drawn from the solution with a pipette and stained with 10 μl of Turk's solution for 10 min. The Neubauer chamber (Rohem Industries Private Limited, India) was then charged with the stained cell suspension and the neutrophils were counted.

Statistical analysis

The data were collected and tabulated in Microsoft Excel format and the statistical analysis was done using SPSS 18 software (IBM, Armonk, NY, USA) and was expressed as mean ± standard deviation. The total sample size of the study was 55 females. The repeated ANOVA test was used for the comparison of MGI, GBI, and neutrophil counts in the three phases of the menstrual cycle. In addition, post hoc Bonferroni test was conducted for intergroup comparisons of variable in different phases of the menstrual cycle.

Ethical clearance for jdmimsu_60_19

The Institutional Ethics Committee of Yogita Dental College and Hospital approved the study with Ref no YDCH/ETHICS/ 1081/2016 on 2nd Jan 2017.

  Results Top

Comparative analysis of GBI and MGI was performed in the three phases of the menstrual cycle (OP, menstrual phase [MP], and PMP). The mean GBI and mean MGI were found to be highest in the OP followed by menstruation phase and PMP [Table 1] and [Table 2]. The mean neutrophil count was found to be highest in OP followed by PMP and menstruation phase [Table 3]. There were statistically significant differences in GBI, MGI, and neutrophil count among the three phases of the menstrual cycle (P < 0.001) [Table 4], [Table 5], [Table 6].
Table 1: Comparing the gingival bleeding index at various intervals of time (ovulatory phase, menstrual phase, and premenstruation phase)

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Table 2: Comparing the modified gingival index at various intervals of time (ovulatory phase, menstrual phase, and premenstruation phase)

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Table 3: Comparing neutrophil count at various interval of time (ovulatory phase, menstrual phase, and premenstruation phase)

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Table 4: Comparison of the gingival bleeding index between different phases of the menstrual cycle

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Table 5: Comparison of the modified gingival index in between different phases of the menstrual cycle

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Table 6: Comparison of neutrophil count in different phases of the menstrual cycle

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  Discussion Top

This study investigated the effect of the menstrual cycle on the clinical parameters such as MGI and GBI and the neutrophil count in GCF in periodontally healthy females at the three different phases of the menstrual cycle.

The results of the present study showed significant changes in MGI and GBI in the OP, MH, and PMP phase of the menstrual cycle, with both the clinical parameters MGI and GBI being highest in the OP phase of the menstrual cycle. This was in accordance with the study conducted by Machtei et al.[21] and Kawamoto et al.,[29] who found an increase in MGI in the OP and PMP of the menstrual cycle, attributing it to the fluctuations in sex hormones such as estrogen and progesterone. Furthermore, the results of the present study are in favor of the study by Khosravisamani et al. who showed higher GBI in the OP compared with the MP phase.[26]

Peak progesterone levels are observed during the PMP phase of the menstrual cycle and increased levels of progesterone lead to an increase in the permeability of blood vessels.[17] Based on this fact, the highest GBI and MGI values are seen during the peak progesterone levels (i.e., in the PMP phase).[30] However, a contrary relationship was observed between the GBI and MGI in the present study.

Andersen and Cimasoni in 1993 described the widely accepted method for neutrophil collection.[19] This method involves the collection of GCF using Millipore filter paper and subsequent analysis of the neutrophil count in the GCF collected on the filter paper. There are numerous ways of collecting GCF for neutrophil count analysis. This method is preferred as it overcomes the shortcomings of other methods such as the clumping of cells in the Styrolux paper and inaccurate determination of the dilution factor in the washing method.[31],[32],[33] Calonius stated that the normal salivary leukocyte count ranges from 110 to 1364 cells/1 mm3 to 770–11,896 cells/1 mm3 from dentulous healthy individuals to inflammatory periodontal conditions.[34]

In the present study, the highest neutrophil count in GCF was observed during the OP followed by PMP. The increase in the levels of estrogen shows two peaks, the first peak being observed just before the OP and the second peak being observed in the luteal phase. Estrogen causes cellular proliferation of blood vessels, stimulates polymorphonuclear leukocyte (PMNL) phagocytosis, inhibits PMNL chemotaxis, and increases the amount of gingival inflammation without any significant increase in plaque.[17] This could explain the increase in neutrophil count in the present study, as during the OP, the estrogen level drops down to the optimum level from its peak level. These results are in favor with the study conducted by Wadhwani and Shetty that evaluated the total leukocyte count in saliva during different phases of the menstrual cycle and concluded that the leukocyte count was at its peak in the OP followed by follicular phase [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5].[35]
Figure 1: Armamentarium used in the present study

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Figure 2: Collection of gingival crevicular fluid from the gingival crevice of the first maxillary molar

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Figure 3: Addition of filter paper strips into the solution containing 40 μl phosphate-buffered saline without calcium, and 3 mm ethylenediaminetetraacetic acid was mixed with 1% bovine serum albumin

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Figure 4: Charging the Neubauer's chamber with cell solution for the assessment of neutrophil count

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Figure 5: Neutrophil seen under ×40 on Neubauer's chamber

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  Conclusion Top

However, more investigations are still needed to establish the cause of inflammation in the periodontium during different phases of the menstrual cycle considering the activity of mucosal immune cells and the effect of endogenous steroids on the function of these cells. The results of the present study indicate that every phase of the menstrual cycle influences the periodontium by inducing inflammatory conditions in the periodontium during the different phases of the menstrual cycle.

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Conflicts of intersest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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