CD4 Cells and Primary Immune Cells as Veritable Factors on Reproductive Hormones and Fertility

Introduction

Author’s interest on immunology as a feasible or veritable factor in fertility dated back to Anyiwo, et al., (1983) in a lecture in immunology on an M.Sc. degree class [1]. It was then asserted that “speculation was in vogue that infertility could also arise from incompatibility between the sperms and the ova.” In an understanding from Garcia Velasco (2017) and Bashiri, et al., (2017) that fact had been confirmed, and also explained part-failures in In Vitro Fertilization (IVF) [2,3].

Recently, it is now known, immunologically, that a father and a mother have different parental Major Histocompatibility Complex (MHC) factor, as well as different Human Leucocyte Antigens (HLA). Further, a foetus is half a factor from a father (spermatozoa) and half a factor from a mother (ovum); consequently, a foetus should hence be expected to have different MHC and HLA from and implantation mother. Indeed, immune system of embryos is different from that of the pregnant woman, as it as well contains genes from the father as stated above, which are strange to the immune system of the mother. Therefore, immunologically, the mother should regard the foetus as an antigen, to which her immune components will attack and destroys. But this is always not so; because, the Regulatory T cells (T Reg) of the mother creates immunologically tolerance state between the mother and the foetus, so the foetus stays, grows and mature. The T-Reg are CD4 and CD25.

In a more definite explanation by Robertson, et al., (2018), the immune system of embryos is different from that of the pregnant woman, as it contains genes from the father as well, which are unknown to the immune system of the mother [4]. For a pregnancy to be normal, the woman’s immune system develops a mechanism of immune tolerance in order not to attack the embryo. One of the most important immune cells is the lymphocytes or white blood cells, capable of recognizing their own structures and also of producing antibodies that recognize foreign substances.

In actual fact, it is the embryo itself that “warns” the pregnant woman through the expression of the HLA-G Antigen, which function is to erase the cells of the immune system in order for the embryo to continue growing in the womb Shushan and Schenker (1992) [5].

Justification of the Research

Role of immunology in fertility is not well-known by many medicos; to date, some still reflects only on reproductive hormones to investigate infertility. This usually ends up in enigma, especially in some cases of persistent infertility in situations of apparent adequate hormonal balances. Such unexplained or idiopathic infertility is a condition in which couples are not able to conceive without any definite causes Parais et al., (2022) [6].

It was on premise of this known role of T-Reg, and on a tripod that immune reactions are interplays of many combined factors parri passu fertility hormones, that the authors sort to investigate the direct relationship of CD4 and other primary immune cells on cases of infertility.

Aim and Objectives

Aim

The aim of this research is to investigate the role of CD4 and primary immune cells as veritable factors on reproductive hormones and fertility.

Specific Objectives

1. Select patients on medical doctors’ provisional diagnosis of infertility.

2. Collect blood samples from them.

3. Analyze each of the blood samples for the levels of:

a. CD4 using a Partec Flow Cytometer.

b. The five primary immune cells using Mindray BC-5150 Fully Automated Blood Cells Analyzer.

c. The six fertility hormones (Follicle Stimulating hormone, Luteinizing hormone, Prolactin, Progesterone, Testosterone and Estradiol), using I-ChromaTM Reader made by Boditech Med Inc.

4. Analyzing the result using Paired Students t-test at 5% (0.05) alpha or significant level; and p value of test statistics used for confirmation.

Null Hypothesis

Ho1: Effect of hormones and CD4 are in equilibrium

Ho2: Effect of hormones and primary immune cells are in equilibrium

Materials and Methods

Collection of Blood Samples for Analysis

The patient’s sleeve was raised above the left elbow, and a tourniquet tied to the upper arm. With the patient’s fist clenched, the area where the needle will be inserted was swabbed with methylated spirit soaked in a cotton wool, then the cover of the hypodermic needle was removed without touching the needle tip, then the now sterilely opened needle, in a slanting position, was gently inserted into one of the most prominently displayed veins in the arm. Then the syringe was gently drawn up to suck in the blood. After about 10ml of blood sucked, the tourniquet was loosened, and the hypodermic needle gently withdrawn from the patient’s arm. The blood sample were then evenly share into three different respective bottles for the hormonal assays, the CD4 counts and the full blood counts. The methylated spirit soaked in a cotton wool was also used to cover the point of insertion of the needle for few minutes to control bleeding.

Analysis pf Primary Immune Cells

The primary immune cells analysis was done with Mindray BC- 5150 Fully Automated Blood Cells Analyzer. 2.5ml of the venous blood collected in an EDTA bottle was placed in a mixer until ready for use. The haematology auto blood analyzer was put on. Then the probe of the analyzer was inserted into the bottle with the blood sample to allow the analyzer to pick the well-mixed blood sample. The result is displayed on the autoanalyzer’s screen, read, printed out and recorded.

CD4 Cells Count

Principle Of Flow Cytometry: The basic principle of flow cytometry is the passage of cells in a single file in front of a laser so they can be detected, counted and sorted. Cell components (such as CD4+ TEST cells) are labelled and then excited by the laser to emit light at varying wavelengths.

Procedure: The CD4 cells count was done with Partec Flow Cytometer Code No. CY-S-3022. The instrument main power was switched on at the back of the instrument and then the green button was pushed on the left of the instrument.

Cleaning: Sample tube was first plugged with cleaning solution and inserted into the sample port, then the botton of the flow cytometer was pressed to start the measurement; after the measurement had stopped automatically, another sample tube containing a decontamination solution was plugged into sample port. When the cleaning procedure had stopped automatically the process was repeated with 1.6ml of sheath fluid in order to remove residual cleaning solution.

Quality Control (count check beads green): A sample tube with well mixed 850μl count check beads green was plugged into the sample port and the start button pressed to begin measurement. When the measurement and cleaning procedure have stopped automatically, the result is indicated at the result area on the sceen This was compared with the lot specific number and check if it was within the allowed 10% range.

Sample preparation for Absolute CD4 count (wet): 20μL of antibody m Ab PE was pippeted into partec tube. 20μL of whole blood was then added into the tube containing the antibody. This was mixed gently and incubated at 15 minutes in a dark field. Then 800μL of no lyse buffer solution was added and analyzed with the C.Y flow counter. For the analysis, the script for CD4 measurement was loaded. Then sample tube with the prepared blood sample was inserted into the machine. Before measurement, the gain value and gating for proper CD4 T-cell measurement is selected. Then measurement started. After the measurement, the machine cleans automatically. The CD4 Count result is then displayed on the screen, read, printed out and recorded

Reproductive hormones analysis

The six reproductive hormones (Follicle Stimulating hormone, Luteinizing hormone, Prolactin, Progesterone, Testosterone and Estradiol) were analysed with I-ChromaTM Reader made by Boditech Med Inc.

Procedures:

a) Luteinizing Hormone: 150uL of serum was transferred to the detection buffer. The tube was covered and then mixed by shaking about 10 times. 75uL of the mixture was then loaded into the sample well on the cartridge, incubated at room temperature for 15 minutes, and scanned using the I-Chroma scanner. The results were displayed on the screen, read and recorded.

b) Follicle Stimulating Hormone: Same as in the Luteinizing hormone.

c) Prolactin: 150uL of diluent was added to the granules on the sample tube, then 75uL of the blood sample was added to the sample tube, incubated at room temperature for 10minutes, and scanned using the I-Chroma scanner. The results were displayed on the sceen of the scanner, read and recorded.

d) Progesterone: 150uL diluent was added to the granules on the sample tube, then 30uL of the blood sample was added to the sample tube, mixed by shaking for about 10minutes. 75uL of the mixture was pipetted into the sample well on the cartridge, incubated at room temperature for 15 minutes, then scanned using the I-Chroma scanner. The results were displayed on the sceen, red and recorded.

e) Testosterone: 30uL displacing reagent was added to a tube. 75uL of the blood sample was added to the displacing reagent, mixed and incubated for 3minutes. 75uL of to the mixture was transferred to the detection buffer tube. Mixed again. 75uL of this mixture was put into the sample well on the cartridge, incubated the loaded cartridge at room temperature for 12 minutes, then scsnned on the I-Chroma scanner. The results were displayed on the I-Chroma scanner screen, read and recorded.

Statistics

The results were analysized using Paired Students t-test at 5% (0.05) alpha or significant level; and p value of test statistics used for confirmation.

Results

Table 1: Impact of CD4 Cells on the Reproductive Hormones.

Note*: Key: FSH=Follicles Stimulating Hormone, LH=Luteinizing Hormone, PRL=Prolactin, EZ=Estradiol, PROG=Progesterone, TESTO=Testosterone, CD4=Cluster of Differentiation, AM 1-AM 7=Patients, RR=Reference Range.

Table 1 showed that all the patients have low CD4 counts (Range: 157-490). Our hypothesis (Ho1) was rejected: (i.e., Yes) that CD4 cells have impact on the reproductive hormones; p values confirmed negative correlative association between CD4 and the six hormones (Table 1).

Table 2 showed that our hypotheses was rejected: (i.e., Yes), PIC have impact on the reproductive hormones. Table 2 also showed that all the PIC have impact on PRL. Except on EZ. Neutrophil (N) has very significant impact on all the hormones. Except on FSH. Lymphocytes (L) has very significant impact on all the hormones. Only on PRL that all the PIC have impact. Except on PRL & TESTO, WBC (T) has no significant impact on the hormones. Only N & L have significant impact on the PROG. Only WBC (T), N & L have impact on TESTO. Except L none of the PIC has impact on the EZ. Monocyte, Eosinophil & Basophils has impact only on FSH, LH & PLT (Table 2).

Table 2: Impact of Primary Immune Cells on the Reproductive Hormones.

Note*: Key: FSH=Follicles Stimulating Hormone, LH=Luteinizing hormone, PRL=Prolactin, EZ=Estradiol, PROG=Progesterone, TESTO=Testosterone, CD4=Cluster of Differentiation, WBC (T)=White Blood Cells (Total Count), N=Neutrophil, L=Lymphocytes, M=Monocytes, E=Eosinophils, B=Basophils.

Table 3 showed that the neutrophil counts were below the reference range in the Mean and in all the patients, except Patient Number 7 which was above the reference range. Table 3 also showed that the lymphocyte counts were above the reference range in the Mean, and in all the patients, except Patient Number 7 which is within the reference range. WBC (T) were all within the reference range except in Patient number 1 (Table 3).

Table 3: Impact of Primary Immune Cells on the Reproductive Hormones.

Note*: Key: N=Neutrophil, L=Lymphocytes, M=Monocytes, E=Eosinophils, B=Basophils, WBC (T)=White blood cells (Total), 1-7=Specimens, R/R.=Reference Range.

Discussion

Role of immunology in fertility is not well-known by all medicos; to date, many still concentrate only on reproductive hormones to investigate infertility Robertson, et al., (2018) [4]. This may end up in enigma, especially in cases of persistent infertility in situations of apparent adequate hormonal balances, as was seen in this work Table 1. This is as a result of their basal knowledge in immunology, immunopathology, immunotherapy and immunodiagnosis/ immunochemistry or immunoassay.

Regulatory T cells or Treg cells (i.e., CD4+ CD25+), have recently been implicated in human pregnancy as key players in protecting the conceptus from alloreactive immune rejection Robertson, et al., (2018) [4]. An increase in circulating Treg cells is evident in pregnancy from the first trimester until shortly after delivery Ciraci et al., (2019) [7]. From the low CD4 counts obtained in this work Table 1 and compared with the adequate hormonal balances of all the patients, the cause of infertility is obviously immunologic infertility. Besides, our hypotheses were largely rejected: (i.e., Yes) that CD4 cells (Ho1) and PIC (Ho2) have impact on the hormones Tables 1,2; and p values confirmed negative correlative association between CD4 & the six hormone’s Table 1.

Regulatory T cells are specialized subpopulation of T-lymphocytes that act to suppress immune response, thereby maintaining homeostasis and self-tolerance. Further, it has been shown that T Reg are able to inhibit T cell proliferation and cytokine production as well as play critical role in preventing autoimmunity. That is the same way it creates an active state of maternal immune tolerance (e.g., of spermatozoa and foetus), thereby ensure a robust placenta and sustain pregnancy.

Thus, pregnancy is immunologically re-defined as a special, exceptional situation in a woman’s body, as it is forced to be home for a “foreign body” for 9months Paraiso, et al., (2022) [6]. This is against the formal definition as a failure to conceive after a year of unprotected intercourse with the same partner. Dysregulation in T Reg cell frequency [in number, as was seen in this work Table 1 and consequently in functions may lead to the development of autoimmunity, as well as immune imbalance or tolerance between the implantation mother and spermatozoa/foetus or embryo, as was perceived in this work. – result of which would be maternal intolerance of foetus/spermatozoa as cause of inability to conceive. This situation is referred to as “immunologic infertility.”

Hence, besides hormonal imbalance, immunologic infertility greatly contributes to inability to conceive. Consequently, CD4 cells count (by extension as T Reg, CD25 inclusive) should be a paradigm in the investigation of infertility.

Immunological infertility is a bit under-defined by Dondero, et al., (1993, 2011) as the presence, in one or both partners, of an anti-sperm immune reaction capable of interfering with fertility variables; because other immunological situations of infertility exist [8,9]. Indeed, according to Shibahara (2022) and Tung, et al., (2017), a significant number of infertile men show an autoimmunity to sperm, and that experiments have suggested that Anti-Sperm Antibodies (ASA) can interfere with the fertilizing ability of spermatozoa [10,11].

ASA can act negatively on the motility of spermatozoa in semen, on their ability to pass through female genital secretions, or on the penetration of the oocyte. In particular, owing to in vitro fertilization techniques, it has been possible to demonstrate the effects of antibody-bound sperm directly, at the level of in vitro gamete interaction Shibahara, 2022; McLachlan, 2002 [10,12]. Among factors responsible for this form of autoimmunity is the situation referred to as “expression of sequestered or occult or hidden antigens,” (such as the antigens of the testis, eyeballs, ovary, etc,) due to when there is break up of their protected tissue-blood barrier, are thereafter exposed to the body’s immune cells which consequently starts destroying them. Infections and trauma are one the major causes of such break in tissue-blood barrier, for which infertility is the penalty.

The noted percent rise in lymphocyte in this work Table 3 is that they must have come from other conventional lymphocytes ((B-cell, Nk cells and other classes of CD cells) and not from the required CD4+ and CD25+ necessary to maintain immunetolerance to sustain implantation in the mothers; and the deficient CD4 detected from all the patients agreed with the assertion. The neutrophil low count agreed with our rejected Ho2 hypotheses (i.e., Yes), PIC have impact on the reproductive hormones Tables 2,3, and the P value further confirmed the negative correlative association between PIC and the six reproductive hormones Table 3.

Conclusion

In conclusion, under no hormonal abnormality, this work strongly suggests that the only other explanation for the inability to conceive resides on the low CD4 cells (and its consequent deficient immune-regulation), plus statistically significant impacts of it and PIC on hormones; hence, they are veritable factors. CD4 cells counts (and primary immune cells counts) therefore should be a paradigm in investigations of infertility.

Acknowledgement

The authors are grateful to Basil Okoye for the statistical analysis of this research.

Competing Interest

The authors declare no competing or conflicting interest whatsoever in this research.

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