FIZIOLOGIA
physiology
FOUNDING EDITORCHIEF EDITOR
CO-CHIEF EDITORSASSOCIATE EDITORSEXECUTIVE EDITORSFRANCISC SCHNEIDERCARMEN PANAITESCUCARMEN TATUFLORINA BOJINMIHAI NECHIFORSORIN RIGAGABRIELA TANASIEDACIANA NISTORCALIN MUNTEANEDITORIAL BOARD
ARDELEAN AURELBĂDĂRĂU ANCA
BENEDEK GYORGYBENGA GHEORGHECOJOCARU MANOLEGĂLUȘCAN ATENAIANCAU MARIAMIHALAŞ GEORGETA
MUNTEAN DANINAMUREŞAN ADRIANANESTIANU VALERIUOPREA TUDORPANAITESCU CARMEN(Arad)(Bucureşti)(Szeged)(Cluj)(Bucureşti)(Timișoara)(Craiova)(Timişoara)(Timişoara)(Cluj)(Craiova)(New Mexico)(Timişoara)PĂUNESCU VIRGILPETROIU ANAPODARIU ANGELA CODRUTA
RÂCZ OLIVERRIGA DANSABĂU MARIUS
SAULEA I. AURELSIMIONESCU MAIASWYNGHEDAUW BERNARDTANGUAY M. ROBERTTATU ROMULUS FABIANVLAD AURELIANVOICU VICTORZĂGREAN LEON

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CUPRINS
INFECȚIA ȘI COLONIZAREA BACTERIANĂ LA FEMEILE ÎNSĂRCINATE ȘI LA NOU-NĂSCUȚI…………………………….…….4
Raluca-Oana Scarlat, Corina Mușuroi, Delia Muntean, Daliborca Vlad, Monica Licker
INFECȚIILE GENITALE: O CAUZĂ MONDIALĂ A INFERTILITĂȚII FEMININE…………………………………………...................12
Raluca-Oana Scarlat, Vlad Daliborca, Monica Licker
MODIFICARI ALE UNOR COMPONENTE ALE ERITROCITELOR IN DIABETUL ZAHARAT……………………………………….17
Matei Daniela Mariana
POSIBILE MODIFICĂRI FIZIOLOGICE INDUSE DE PRACTICA RUGĂCIUNII ÎN TRADIȚIA CREȘTIN-ORTODOXĂ……….….28
Adrian Sorin Mihalache, Ana-Maria Zăgrean, Daniela Bălan, Iulian Stoleriu, Leon Zăgrean
METODE DE DIAGNOSTIC DE PRECIZIE ÎN TUMORILE SOLIDE – SECVENȚIEREA DE GENERAȚIE URMĂTOARE (NGS).47
Octavia-Oana Harich, Simona Anghel, Carmen Tatu, Gabriela Tănasie, Virgil Păunescu, Carmen Panaitescu

CONTENTSMICROBIAL INFECTION AND COLONIZATION IN PREGNANT WOMEN AND NEWBORNS.......................................................4
Raluca-Oana Scarlat, Corina Mușuroi, Delia Muntean, Daliborca Vlad, Monica Licker
GENITAL TRACT INFECTIONS: A WORLDWIDE CAUSE OF FEMALE INFERTILITY………………………………………………12
Raluca-Oana Scarlat, Vlad Daliborca, Monica Licker
ALTERATION OF SOME RED BLOOD CELL COMPONENTS IN DIABETES MELLITUS…………………………………………...17
Matei Daniela Mariana
POSSIBLE PHYSIOLOGICAL CHANGES INDUCED BY THE PRACTICE OF PRAYER IN THE CHRISTIAN-ORTHODOX
TRADITION……………………………………………………………..………………...........................................................................28
Adrian Sorin Mihalache, Ana-Maria Zăgrean, Daniela Bălan, Iulian Stoleriu, Leon Zăgrean
PRECISION DIAGNOSTIC METHODS IN SOLID TUMORS – NEXT GENERATION SEQUENCING (NGS)……………………….47
Octavia-Oana Harich, Simona Anghel, Carmen Tatu, Gabriela Tănasie, Virgil Păunescu, Carmen Panaitescu

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MICROBIAL INFECTION AND COLONIZATIONPREGNANT WOMEN AND NEWBORNSINRALUCA-OANA SCARLAT1, CORINA MUȘUROI1,2, DELIA MUNTEAN1,2, DALIBORCA VLAD1,3,
MONICA LICKER1,2
1.Clinical laboratory, “Pius Brînzeu” Clinical County Emergency Hospital, Timișoara
2.Multidisciplinary Centre for Antibiotic Resistance Research (MULTI-REZ), “Victor Babeș” University of Medicine and Pharmacy Timișoara
3.Pharmacology Department, “Victor Babeș” University of Medicine and Pharmacy Timișoara

ABSTRACT
Different types of microorganisms, including bacteria, parasites, viruses and fungi, can colonize and infect the genitourinary tract of the female.
Moreover, not few of the female patients exposed to bacterial genital infections get pregnant, increasing significantly the risk of transmission totheir fetuses. Neonatal infections are of great importance due to their high morbidity and mortality. The aim of the present study was to identify
the bacterial strains from cultures of cervical samples collected from pregnant women during one-year period, to determine the prevalence andthe microbial resistance of each pathogen and to correlate the results with different stages of pregnancy or with various pregnancy pathologies.
Data from the present study involving cervical samples has revealed the following: the most prevalent Gram-negative pathogen was E. coli
whereas group B streptococci (GBS) presented the highest prevalence amongst the Gram-positive bacteria. Importantly, the outcomes haveshown a substantial number of microorganisms detected in cultures from samples of pregnant women with pregnancy in evolution/full-term birth.
However, not few pathogens were identified from cultures of pregnant women presenting an abortion or other pregnancy pathologies, somestrains exhibiting microbial resistance that should rise attention when it comes to provide health care and treatment for the pregnant women
during pregnancy or labor. Expectantly, the acquisition and implementation of a new automatic microbiological sample-processing line may help
with identifying the microorganisms faster and may increase the vigilance on infection surveillance in pregnant women and newborns.
Key words: genital infections, pregnancy, neonatal pathologies, resistance phenotype

INTRODUCTION
To date, many researches had brought to lightimportant information about the human microbiome, themiscellaneous microbial communities that colonize different
sites of the body. Some studies have shown that 44 𝑥 1012
individual microbial cells reside within the female humanbodies, while the male bodies are colonized by 38 𝑥 1012
microbial cells [1]. Although the role of this nonhuman life
community in preserving host functionality and health iscrucial, it still remains incompletely understood. Alterationsin the human microbiota are associated with host infections
and pathologies, such as urinary tract infections (UTIs),
bacterial vaginosis (BV), inflammatory bowel disease (IBD)and various metabolic diseases. Widely, uro genital tract
infections (UGTIs) are classified into a) UTIs which include
cystitis, prostatitis, pyelonephritis and b) genital tract
infections such as cervicitis, urethritis, epididymitis,
endometritis and pelvic inflammatory disease (PID) [2].
There are many agents that are able to interfere with theurinary tract (UT) causing UTIs, including pathogens suchas bacteria, fungi and viruses [2]. Over the years, theresearchers have published a small amount of data
concerning modifications in the causative agents’ frequency
in patients with community-acquired UTIs, contrasting the4

etiology of hospital- acquired UTIs which have suffered
substantial changes.
Enteric bacteria (especially,
Escherichia coli) demonstrated their pathogenicity andimplication in the etiology of UTIs over time, remaining themost common cause of UTI in outpatients. Regarding the
hospital-acquired UTI, some important studies had revealed
that infections with Escherichia coli (E. coli) and Proteus
species are decreasing, whilst those caused by Klebsiellapneumoniae (K. pneumoniae), Acinetobacter spp.,
Pseudomonas spp., yeasts and group B streptococci (GBS)
are gaining ground [3]. Microbiologically, in the majority offungal UTIs, Candida albicans is found as the causative
pathogen, counting a percentage of 50% to 70% of isolates,
followed by Candida glabrata, Candida tropicalis, Candida
parapsilosis and Candida krusei [4].
When it comes to the reproductive function of females,a variety of microorganisms are involved in genital tract
infections. Not a few infections of the genitalia are causedby sexually-transmitted pathogens [2]. Chlamydia
trachomatis is the most important bacterial cause of PID,
affecting predominantly young women [5]. Neisseria
gonorrhoeae represents the well-known bacterial pathogen
responsible for the sexually-transmitted disease (STD),
gonorrhea [6]. A substantial body of clinical literature

Fiziologia - Physiology • 2023.1(104)including experimental inoculations in humans and animals
link Gardnerella vaginalis to vaginal dysbiosis, also named
bacterial vaginosis (BV) [7]. Besides of vaginal dysbiosis,
Ureaplasma urealyticum and Mycoplasma hominis are
considered causes of endometritis, PID, spontaneousabortion, preterm delivery, infertility and pyelonephritis [8].
Treponema pallidum is widely known as the responsible
microorganism for syphilis, causing inflammation,
obstruction of the fallopian tube and endometritis [9].
Trichomonas vaginalis is the most prevalent parasite with asignificant role in female genital tract infections andunfavorable pregnancy outcomes [10].
During pregnancy, various species of bacteria may
colonize and infect the vagina, resulting in preterm labor,premature rupture of the membranes, amniotic fluid
infections and low birth weight. Not few studies reveal thevertical transmission of bacteria from pregnant women to
their unborn babies, frequently leading to obstetric and fetal
infections [11].
MATERIALS AND METHODS
An institution-based cross-sectional study was
conducted on patients admitted at the “Pius Brînzeu” County
Clinical Emergency Hospital Timișoara (SCJUPBT),
Romania. It is a tertiary medical care unit with 1174 beds, ofwhich 82 beds are in Obstetrics-Gynecology (OG)
Departments.
During one-year period, between September 1st,
2021 and September 1st, 2022, from a total number of 506
female patients hospitalized in OG Departments, 260(51.38%) were pregnant women and 246 (48.62%)
presented gynecological pathology. All the strains includedin the study were identified in cervical and non-cervical
specimens and were collected from the pregnant women
hospitalized in the clinic due to any infectious disease. The
260 women under medical follow-up were monitored for: 1)
maternal diseases such as hypertension, kidney diseases,
thyroiditis, anemia, etc. and 2) maternal diseases associatedwith pregnancy including preeclampsia, pathologicalpregnancy and placenta praevia.
The microbial identification was facilitated by theuse of Matrix-Assisted Laser Desorption Ionization Time-OfFlight Mass Spectrometry (MALDI-TOF MS). Compared tothe older, conventional techniques, this high-tech device canaccurately and promptly identify yeast, molds, as well as
various genus and species of bacteria.
Antimicrobial sensitivity tests were performed using
disk diffusion and the VITEK 2 automated system(bioMérieux Marcy l’Etoile, France). The VITEK-2 system’sinterpretation of the antimicrobial susceptibility data wasmade using the minimum inhibitory concentration (MIC)
accordingly to the CLSI standards, 2021.
In one case of a newborn with bloodstream infection(BSI), the antimicrobial-resistance (AMR) genes wereidentified with Unyvero, a complex system that uses
multiplex PCR technology that concomitantly identifies the

Fiziologia - Physiology • 2023.1(104)

microorganisms and the antibiotic resistance markers.
The present study was approved by the decision of the
Ethics Commission of SCJUPBT no 346/31.10.2022.
RESULTS
From the total of 260 (51.38%) pregnant women
included in this study, a number of 275 collected samples
were examined. Furthermore, the samples were divided into
two groups as it follows: 1) cervical specimens and 2) clinicalspecimens other than cervical (urine cultures, amniotic fluids,
peritoneal fluids, wound secretions and sputum). The
cervical samples counted a number of 250 and the rest of
the 25 represented samples from different sites, excluding
cervical. For 80 of the pregnancies, cesarean delivery (Csection) was performed. Only 6 (7.5%) of the C-sections
were problematic.
In the first group involving the 250 cervical samples
from pregnant women, 265 strains of bacteria were identified.
Furthermore, the patients from whom the colonized/infectedcervical samples were collected were, on one hand,pregnant women with healthy pregnancies in different
stages and on the other hand, expectant mothers
hospitalized in order to receive medical care and treatment
for several pathologies occurred in the evolution of thepregnancy: abortion, premature birth and ectopic pregnancy.

5

Firstly, the identified strains were classified. Amongthe 265 strains, minimum 4 distinct species of bacteria weredetected: 96 (36.23%) strains of E. coli, 80 (30.19%) of GBS,
49 (18.5%) of S. aureus, 32 (12.07%) of K. pneumoniae anda small number, 8 (3.01%), of other bacteria. Moreover,these various strains were isolated from patients withpregnancy-related pathologies (Table I) and with pregnancy
in evolution/full-term birth (Table II). Importantly toemphasize is that there were more isolates identified in
distinct samples of the same patient.
Secondly, in order to identify and correlate thepotential risk of infection in newborns with natural birth, thenumber of patients with monitored pregnancy, pregnancy
pathologies and the respective microorganisms were
determined. Amongst the patients with pregnancy inevolution/full-term birth, from a total of 174 pregnant women,
60 (35.08%) presented GBS strains, 48 (28.07%) E. coli, 37(21.63%) S. aureus, 21 (12.28%) K. pneumoniae and 8(4.67%) other bacteria (Table III). Regarding the patients
who underwent an abortion, from a total of 57 pregnant
women, 33 (57.89%) were found with E. coli, 12 (21.05%)
with GBS, 6 (10.52%) with S. aureus, 6 (10.52%) with K.
pneumoniae and none (0%) with other bacteria. The
samples from patients who gave birth to premature babies
counted a number of 21, of which 8 (38.09%) were E. coli
strains, 5 (23.8%) were S. aureus, 4 (19.04%) GBS, others
4 (19.04%) K. pneumoniae and 0 (0%) other bacteria. Fromthe patients presenting ectopic pregnancies, 13 samples
were collected. Among these pregnant women, 7 (53.84%)
were positive for E. coli, 4 (30.76%) of them for GBS, 1(7.69%) for S. aureus, another 1 (7.69%) for K. pneumoniae
and none (0%) for other bacteria (Table III). After the numberof patients and the isolates were determined, a comparative
presentation was performed (Fig 1).
Concerning the AMR phenotypes, in the case of E.
coli strains were found 3 resistance rates as it follows: apercentage of 8.33% resistance to aminoglycosides (RAGs), 8.33% to fluoroquinolones (R-FQs) and 23% tosulfamethoxazole-trimethoprim (R-SXT).
Regarding the GBS, 49 (61.25%) strains were tetracyclineresistant (R-TE), 22 (27.16%) were multi-drug-resistant(MDR), 20 (25%) presented macrolide-lincosamidestreptogramin B (MLSB) inducible resistance, 9 (11.25%)
had R-FQs and 6 (7.5%) had β-lactamase (BL) resistancephenotype. Among the strains of S. aureus, 22 (44.89%) had
resistance to BL, 19 (38.77%) were MDR strains, 15(30.61%) were MRSA, 13 (26.53%) had MLSB phenotype,
13 (26.53%) were resistant to macrolides (R-M), 5 (10.2%)
were FQ-R and 5 (10.2%) were SXT-R. K. pneumoniae
strains presented the following resistance patterns: 5(15.62%) SXT-R, 2 (6.25%) BL resistance, 2 (6.25%) FQ-R,
1 (3.12%) AG-R and another 1 (3.12%) MDR pattern.
Regarding the second group, various types of
microorganisms were detected in each of the samples,
excluding cervical samples. In the urine cultures, a total of
15 microorganisms were6

identified: 13 (86.6%) of E. coli, 1 (6.7%) of GBS and 1 (6.7%)
of Enterococcus spp. Moreover, among the E. coli strains, 1
resistance phenotype of extended-spectrum β-lactamase(ESBL)-producing was ascertained.

Fig. 1. The percentage of each microorganism
isolated from pregnant women in different stages ofthe pregnancy or isolated from women with various
pregnancy pathologies

The pregnant women with positive urine cultures werediagnosed with UTIs. Of them, 10 were 27-32 weeks
pregnant and 5 had a full-term pregnancy. The amniotic
fluids samples were positive for the following
microorganisms: 1 for Staphylococcus epidermidis, 1 for
Staphylococcus lugdunensis and 1 for Enterococcus
faecalis. None of these 3 presented any resistancephenotypes. From the 2 peritoneal fluid specimens, 3
microorganisms were isolated: from the first, Pseudomonas
aeruginosa and Acinetobacter baumannii and from thesecond, S. aureus. When referring to the AMR patterns,
carbapenems-resistance (CR) and sulfonamides resistancephenotypes were found in the case of Acinetobacterbaumannii strain, while BL-producing phenotype was foundin the case of S. aureus. Additionally, with the help ofUnyvero system, 3 resistance genes were detected in thecase of Acinetobacter baumannii strain: NDM, OXA-24 andSUL1. Both peritoneal fluid samples were collected frompatients with post-cesarean complications subsequently
solved by total hysterectomy. In the wound culture samples
included in the study, 2 pathogens were identified: 2 samples
with S. aureus and 2 samples with Klebsiella spp. BLproducing resistance phenotype was present in one of thecases with S. aureus and MLSB inducible resistancephenotype was present in the second case. Among thesamples with Klebsiella spp. strains, one was found withcephalosporinase resistance phenotype. Both pathogens
infected the wounds after cesarean section in patients withpre-term births (26-30 weeks) caused by premature ruptureof membranes. A single female patient, diagnosed withsevere acute respiratory syndrome Coronavirus 2 (SARSCoV-2) infection, was positive for Candida albicans in the
sputum. No pattern of resistance was detected.
DISCUSSIONS
During pregnancy, various types of microorganisms
can colonize and infect the genital tract of pregnant women,

Fiziologia - Physiology • 2023.1(104)increasing the chances for the newborn’s health to becompromised. Many pregnant women undergoing genitallarge area of damaged, vulnerable uterine tissue is exposed
to infection, sometimes resulting in puerperal sepsis(childhed fever). Infections with viral pathogens in pregnant
women (rubella, cytomegalovirus-CMV, varicella-zoster
virus-VZV) can cause limb deformities and other severe
lesions to the foetuses and neonates, depending on thegestational age. Bacterial infections originating from thevagina and perineum are more important, occurring
especially when foetal membranes have been ruptured formore than 1-2 days. Infants with very-low birth weights(<1500 grams) tend to be more susceptible to theconsequences of bacterial infections, frequently fatal unlesstreated [12].
The occurrence of E. coli infections still remains oneof the most important challenges in advanced medicine,leading to neonatal septicemia and respiratory tract
infections [13]. In our research, from a total of 265 samples
from pregnant women, 96 (36.23%) were positive for E. coli
in cervical samples and 13 (86.6%) in samples other thancervical (a total of 15), making this bacterial pathogen themost prevalent in this study. Among pregnant women, theprevalence of E. coli differs from country to country.
Regarding the cervical samples, studies from otherparts of the world have shown comparable results. A study
from Central Uganda identified E. coli as the most common
bacterial isolate from pregnant women in a proportion of 34.5%[14]. In India in 2017, very much alike results were
determined: 38.8% E. coli strains [15]. Another study from2021 conducted in Ethiopia came out with similar results of
29.9% E. coli strains among pregnant women [11]. In
contrast with these, a study from Spain identified 15% E. coli
strains in two different types of genital samples: vaginal andendocervical. Although the prevalence was lower incomparison with our study, the detected strains weresignificant because of their virulence factors that permit
vaginal and endocervical colonization, possibly leading to an
early-onset neonatal sepsis (EOS), a potentially fatal
neonatal complication [16]. A similar study from West Africa
reported a percentage of 10.9% E. coli strains identified invaginal and endocervical samples from pregnant women[17]. In our study, E. coli was present with a high prevalence,but taking into account the significance of its presence in thecervix (possible contamination), as well as the low
antimicrobial resistance (indicating community-acquired
strains), it can be claimed that the treatment should beapplied only when the patient’s clinical status requires it.
Relating to the samples other than cervical, E. coli
was the predominant pathogen (86.6%) detected in thepositive urine cultures. Substantial growth was reported in acomparable study from 2022, showing 76.7% E. coli strainsin the urine cultures of pregnant patients [18]. As alreadyacknowledged in the enormous body of the literature, theoutcomes of the neonates corresponding to the pregnant

Fiziologia - Physiology • 2023.1(104)

infections expose their foetuses and neonates to severe
injuries during or after delivery. After delivery (or abortion) a
women affected by UTIs can be very unfavorable. Low/Verylow birth weight, premature birth and perinatal mortality havebeen described [19]. GBS and E. coli are the mostcommonly bacterial pathogens associated with EOS. E. coli
was registered in approximately 24% of all EOS, 81% ofcases involving premature infants [20]. Noteworthy, in thisstudy have been detected 10 pregnant women with pre-term
pregnancies (27-32 weeks) and the 5 with full-term births,
results that may support the previous associations between
pregnancy, UTIs and the negative prognostic in newborns. A
recent case report of an E. coli-transmitted infection
including a pregnant woman and her pre-term twins has
shown a dramatic EOS of the first twin, with respiratory
distress followed by hemodynamic instability. Peculiarly, thesecond twin was clinically well until day 11th of life when his
right scrotum became swollen and tender, being afterwards
diagnosed and successfully treated for scrotal abscess. It isof a great importance for the healthcare practitioners to beaware of the diversity of E. coli clinical presentations and to
immediately act and treat when suspecting EOS [21].
Additionally, neonates with sepsis caused by ESBLproducing E. coli strains represent a group of patients
predisposed to a higher mortality [22]. In this study, one
ESBL-producing E. coli was ascertained in a positive urine
culture. Similar to our result, a fatal outcome of a premature
newborn with meningitis caused by ESBL-producing E. coli
was described recently [22]. These results may support theneed of careful observation of the primal vital signs and thepotential aggressive E. coli infection in newborns.
GBS, also named Streptococcus agalactiae, is a
Gram-positive microorganism that can cause severe
infectious diseases and complications in newborns. A
neonatal GBS infection is frequently acquired from GBScarrying pregnant women by vertical transmission duringlabor or birth (50% of the cases) and in some situations, byhorizontal transmission (other people and the environment)[23]. In pregnant women, GBS can increase the risk of UTI,
chorioamnionitis and sepsis, while among neonates, it canlead to serious complications such as pneumonia,
meningitis, neurological disabilities and neonatal sepsis witha mortality rate of 19.2% in pre-term infant and 2,1% in fullterm infant [23][24]. In this study, GBS presented a highprevalence in patients with pregnancy in evolution or fullterm birth (35.08%) and also in the case of those
hospitalized for pre-term birth (19.04%), results that sustain
the need of a careful screening and an adequate treatment
in order to prevent any sort of neonatal diseases andcomplications. More than this, 61.25% of the GBS isolates
manifested tetracycline-resistance and 27.16% were MDR,
associating resistance to BL, FQ and MLSB. The MDR
strains were identified in cervical secretions of pregnant
women in 7-19 weeks of pregnancy, in the stage of imminent
abortion. Several studies [25-28] reported comparable
patterns of resistance to the same antibiotics that we used7

in the present study, with various differences that may be
due to the country of origin, the region, the race and otherindividual characteristics. Thereby, the strains presenting
significant AMR phenotypes may be problematic andchallenging when treating the patients. Even though
universal screening for GBS colonization including rectovaginal culture swabs has led to an important decrease ofearly-onset disease, there is still an increased number of
infants from GBS negative mothers that contract GBS
strains [24]. Thus, the “MALDI detection with analysis ofpeaks of TOF (MDAPT)” as a novel strategy was suggested
for screening and prevention of serious GBS pathologies innewborns, being able to detect the pathogen with a superiorsensitivity straightly from the culture broth [23].
Another Gram-positive pathogen identified in thisstudy is represented by S. aureus, an important causative
agent of community-acquired and hospital-acquired
infections. An increasing number of severe staphylococcal
infections is nowadays generating difficulties for thehealthcare providers in offering an effective treatment,
especially because of methicillin-resistant S. aureus (MRSA)
strains [29]. The results from the present study haverevealed a percentage of 18.5% S. aureus isolates. In a
study from New York, USA, a similarity was brought to light:
S. aureus was identified with a prevalence of 17.1%, itspresence being significantly associated with GBS
colonization [30]. Contrasting this, numerous studies fromdifferent countries have showed up with lower prevalence ofS. aureus strains as it follows: 14.2% (Pittsburgh, USA) [31],14.5% (Birmingham, USA) [32], 8.2% (Uganda) [14] and15.4% (Africa) [17]. Nevertheless, regarding the detectedstrains of S. aureus, it can be observed that more than ¾isolates were collected from the cultures of the pregnant
women hospitalized for monitoring the pregnancy inevolution or were with full-term birth. Thus, just ¼ of theidentified strains were obtained from the inpatients
presenting abortion or pre-term birth. Despite the fact thatjust a small amount of strains originated from the samples offemales with pregnancy pathologies, the isolates exhibit agreat importance by reason of their multiple AMR
phenotypes. In this way, 44.89% of the isolates showed
resistance to BL, reaching almost half of the total. More
importantly, the MRSA phenotype was ascertained in apercentage of 30.61%, result that is higher than reports fromother studies: 0.5% [33], 0.4% [34] and 0.5% [35]. However,just ¼ of the MRSA strains were obtained from patients
hospitalized for abortion in progress or impending and therest of ¾ had pregnancy in evolution/full-term birth. It is
possible that the overall variations in prevalence may be dueto the sampling error, distinct usage or misuse of antibiotics
and undesirably, due to the poor basic hygiene of the
medical care. Also, an increased rate (38.77%) was
detected pertaining to the MDR strains, finding that may
have considerable public health implications of therespective isolates by restricting the treatment option. This
percentage is comparable with results from a recent study

8(2021), showing 50.9% MDR S. aureus strains [36].
Although not with a crucial role in our study, thesamples from pregnant women obtained from other sites
excluding cervical, including wound culture and peritoneal
fluid culture specimens, revealed 6 of 7 resistant pathogens.
The strains were detected in samples from patients with preterm birth and rupture of membranes and complicated Csections practiced in all of these cases, increasing the riskof side effects of antibiotic usage to the breastfeeding
mothers.
CONCLUSIONSThe colonization and the infection of pregnant
women with various microorganisms (including bacteria,
parasites, viruses, fungus) have been associated withserious life-threating neonatal diseases, including meningitis,
necrotizing enterocolitis and sepsis [37].
In the present study, regarding the Gram-negative
bacilli from the samples of cervical secretion, low incidenceof resistant strains with clinical significance have been
revealed. Among these, E. coli was found as the mostprevalent pathogen but with a low antimicrobial resistance,
indicating its presence as colonization, as well as acquired
from community. However, it is crucial to understand that atreatment in these cases must be applied only if the clinicalcondition of the patient requires it and that their resistancepattern must be known when detecting early infections in
newborns. When it comes to Gram-positive cocci, highfrequencies were registered, presenting different resistancephenotypes that pose therapeutic challenges. GBS strains
provided a high prevalence, followed by S. aureus which isof a great importance due to the multiple antimicrobial
resistance mechanisms. Therefore, adequate supervisionand treatment due to the potential for illness in neonates ismandatory.
To summarize, the newborn infections with MDR
pathogens, especially those caused by ESBL/CR-producing
Enterobacterales or MRSA are correlated with an increased
risk of morbidity and mortality. Unfortunately, the treatment
options for such infections are limited. Currently, efficacy andsafety of novel antibiotics are extrapolated from adult data,requiring an optimization of the dose. The continuing
evolution of clinical trials is revealing promising results using
ceftazidime/avibactam,
meropenem/vaborbactam(Vabormere, Vaborem), imipenem/cilastatin/relebactam(Recarbio) and cefiderocol (Fetroja).
FUTURE DIRECTIONS
The acquisition and implementation of the newautomatic microbiological sample-processing line may comewith helping in shortening times by using advanced
technology (by 1,5 days), bringing cost efficiency in material
and human resources (more is done with less expense) andin improving patients’ health. The implementation of
automatic standardized microbial culturing may increase theperformance of microbial isolation on culture media, leading

Fiziologia - Physiology • 2023.1(104)to a faster identification of microorganisms and reducing tozero the risk of false negative results. Hopefully, it will
provide safety in terms of contamination of medical
personnel and will allow to increase vigilance on infection
surveillance in pregnant or infertile women and newborns.
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10

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37. Krohn, M. A., Thwin, S. S., Rabe, L. K., Brown, Z., & Hillier,
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610. doi:10.1093/infdis/175.3.606

Fiziologia - Physiology • 2023.1(104)E. coli
Samples ofpregnant
women nr.
%
Abortion nr.
%
Pre-term
birth nr.
%
Ectopic
pregnancy nr.
%
Pregnancy inevolution/fullterm birth nr.
%

GBS

S. aureus

K. pneumoniae

Others

96

80

49

32

8

21

36.23
33
57.89
8

30.19
12
21.05
4

18.5
6
10.52
5

12.07
6
10.52
4

3.01
0
0
0

13

38.09
7

19.04
4

23.8
1

19.04
1

0
0

53.84

30.76

7.69

7.69

0

48
28.07

60
35.08

37
21.63

21
12.28

8
4.67

265
57

174

Table III. The number and the percentages of patients, with pregnancy in evolution/full-term birth and pregnancy
pathologies and the identified microorganisms

INFECȚIA ȘI COLONIZAREA BACTERIANĂ LA FEMEILE INSARCINATE ȘI LA NOUNASCUȚIREZUMAT
Diferite tipuri de microorganisme, bacterii, paraziți, virusuri și fungi, pot coloniza și pot determina infecții la nivelul tractului genito-urinar
feminin. Mai mult decât atât, nu puține femei expuse la infecțiile genitale bacteriene rămân insărcinate, crescând astfel semnificativ riscul detransmitere al infecțiilor la nou-nascuți. Infecțiile neonatale au o importanță deosebită datorită riscului crescut de morbiditate și mortalitate.
Scopul studiului de față a fost identificarea tulpinilor bacteriene din culturi de probe cervicale provenite de la paciente însărcinate pe perioada
unui an de zile, determinarea prevalenței și a rezistenței microbiene pentru fiecare patogen și corelarea rezultatelor cu etapele sarcinii,
respectiv cu diferite patologii de sarcină. Datele studiului prezent au evidențiat următoarele: cel mai frecvent patogen Gram-negativ detectata fost E. coli, în timp ce streptococul de grup B a prezentat cea mai mare prevalență din grupul bacteriilor Gram-positive. Important demenționat este faptul că rezultatele au arătat un număr substanțial de microorganisme depistate în culturile pacientelor cu sarcină în
evoluție/sarcină la termen. Cu toate acestea, nu puțini patogeni au fost identificați în culturile pacientelor care au prezentat avort sau alte
patologii de sarcină, unele tulpini având fenotipuri de rezistență care necesită o atenție sporită în ceea ce privește îngrijirea medicală și
tratamentul infecțiilor din timpul sarcinii sau din timpul travaliului la pacientele însărcinate. În mod expectativ, achiziționarea și implementarea
unei noi linii automate de prelucrare microbiologică a probelor ar putea ajuta la identificarea mult mai rapidă a microorganismelor și ar putea
crește vigilența în supravegherea infecțiilor la pacientele însărcinate și la nou-născuți.
Cuvinte cheie: infecții genitale, sarcină, patologii neonatale, fenotip de rezistență

Titlul proiectului: ROHU-339, „Romanian-Hungarian cross-border project with a wider focus on diagnostics related toinfertility, healthy pregnancy and newborn care”, acronim HEALTH-PREGN-RO-HU
Editorul materialului: Spitalul Clinic Județean de Urgență „Pius Brînzeu” Timișoara
Data publicării: aprilie 2023
Acest proiect este cofinanțat de Uniunea Europeană prin Fondul European de Dezvoltare Regională în cadrul Programului Interreg VA România-Ungaria.
Conţinutul acestui material nu reprezintă în mod necesar poziţia oficială a Uniunii Europene.

Fiziologia - Physiology • 2023.1(104)11Parteneriat pentru un viitor mai bun

www.interreg-rohu.eu

GENITAL TRACT INFECTIONS: A WORLDWIDE CAUSEOF FEMALE INFERTILITYRALUCA-OANA SCARLAT1, VLAD DALIBORCA1,2, MONICA LICKER1,3
1: Clinical laboratory, “Pius Brinzeu” Clinical County Emergency Hospital, Timisoara
2: Pharmacology Department ,“Victor Babeș” University of Medicine and Pharmacy Timișoara
3: Multidisciplinary Center for Antibiotic Resistance Research (MULTI-REZ), “Victor Babeș” University of Medicine and Pharmacy Timișoara

ABSTRACTThe infertility represents in almost all the cases a multifactorial disorder, defined as the inability to conceive after one year of sexual intercourse
without any sort of contraception. There are many causes that have shown to affect the female’s fertility, besides the increased age: uterine
pathologies, tubal and ovulatory dysfunctions. Tubo-peritoneal impairments represent the most important group leading to pelvic inflammatory
disease (PID) and infertility. Untreated genital tract infections such as chlamydial, gonococcal and other infections with aerobic or anaerobic
microorganisms are significantly relevant to the uterine and tubal damage, finally resulting in female infertility. The human microbiota proved toplay an essential role in keeping the reproductive tract balanced, mostly due to the presence of particular Lactobacillus species. Studies havedemonstrated that the decrease of Lactobacillus spp. and the overgrowth of other pathogens together with the local inflammation leads to thewell-known disease, bacterial vaginosis (BV), also involved in infertility. The new technological advances and the intensive screening are themajor contributing factors in reducing the incidence of genital tract infections and provide great pregnancy results.
Key words: infertility, pelvic inflammatory disease (PID), tubal infertility, microbiome

INTRODUCTION
Well defined as the failure of conception after
sexual intercourse without contraception for at least 12
months, infertility affects many couples of the reproductiveage (number of childless women in U.S.: from 1.9 million in1982 to 2.8 million in 1995 [1]). Some important changes in
the clinical infertility practice have made it possible to obtain
successful treatments and to increase the number ofpatients seeking medical attention which implies a burden ofcosts both for the patients and for the health care system too[1]. Moreover, the infertility represents not only a disability of
the reproductive female system, but it can lead to different
forms of psychosocial pathologies in the individuals, likestigmatization, economic deprivation, social isolation anddivorces, and in some cases, to violence [2].
An increased reproductive age, a common trend
in the nowadays couples, represents a significant factor for
the involuntary childlessness. For women, the fertility
decreases significantly in the middle to late 30s, meanwhile
for men, the infertility accelerates after 40s. Many otherconditions in women have shown to increase the risk ofinfertility, such as uterine diseases, tubal impairments andovulatory dysfunctions [3]. Between the different causes
responsible for the women’s failure in getting pregnant, tuboperitoneal factors represent a small, but the most importantgroup leading to inferility, counting approximately 25-30% of
the female factors [4]. Tubo-peritoneal impairments usually

12

include both prox „imal and distal tubular diseases, whichappear mostly due to inflammation caused by genital
infections with various microorganisms, resulting in pelvic
inflammatory disease (PID) and long-term sequelae [4].
RESPONSIBLE PATHOGENS
The most frequent bacterias found as causative
factors in upper genital tract infections are Chlamydia
trachomatis (C. trachomatis), Neisseria gonorrhoeae(N.gonorrhoeae) and some anaerobes, while Trichomonas
vaginalis (T. vaginalis) and Candida albicans (C. albicans)are the most prevalent causes in lower genital tract
infections [5]. These microorganisms ascend from theendocervix through the endometrium (endometritis) and to
the fallopian tubes (salpingitis) along the mucosa, causing
infection and inflammation. Clinically, this entire process isdiagnosed as PID, which generally presents asasymptomatic or minimally symptomatic [6]. Because thebacteria are passing through the endometrium and after thatin the fallopian tubes, salpingitis is very often accompanied
by endometritis. It was shown that approximately 70% to 90%
of women with salpingitis had endometritis in the same time,
contrasting the 8% to 33% with endometritis but without
salpingitis [7]. Although endometritis is usually used as amarker for salpingitis and the latter is associated with chronic
pelvic pain, infertility and reccurent PID, there are studies

Fiziologia - Physiology • 2023.1(104)demonstrating the lack of association between endometritis
and infertility in women with BIP. [7].
C. trachomatis infection is one of the most frequent sexuallytransmitted disease (STD) worldwide. It is often the cause ofurethritis, cervicitis, ectopic pregnancy, PID (e.g. salpingitis,
endometritis) and tubal factor infertility (TFI) [8]. Chlamydia
is an intracellular microorganism with a special
developmental cycle of three stages. One life stage proved
to contribute as a major factor to the mechanisms ofresistance to antibiotics [9]. This bacteria possesses theability to transform repeatedly from a resting form
(elementary body) to a replicative form (reticulate body),
increasing the chances to survive in the reproductive tract[8]. An untreated C. trachomatis infection ascending andattaching to the mucosal surfaces leads to the serious
complication, tubal infertility, which depends on theassociation of some factors which are present during thesalpingitis episode, including the severity of inflammationand the number of episodes [5]. In a study conducted byWeström [10] on 415 women with acute PID, 12.8% had
tubal damage after a single episode of infection, 35.5% after
two episodes and 24 women had three of more, while 18
were sterile. He demonstrated that the number of infections
diminished with age: 30.1% of the reinfected women who
were maximum 20 years old contrasted the 13.2% of those
who were over 21 years old [10].
As the second most important preventable
infectious cause of PID and female infertility, but lessprevalent than C. trachomatis, N.gonorrhoeae is recoveredfrom around 30% of Swedish patients presenting with acutesalpingitis [5]. Between the 1970s and 1980s, acutegonococcal PID was found in approximately 40-50% of
patients in regions of the United States, prevalence that
contrasts with the recent findings showing a percentage ofjust 20% [6]. This suggests that the number of cases ofN.gonorrhoeae infection do not appear nowadays with thesame frequency as they did in the past and that there is avariation in prevalence depending on the patients race. In alarge part of the affected women, N.gonorrhoeae is the only
microorganism detected, but neither are differences in theamount of patients presenting with N.gonorrhoeae plus a
combination of aerobic and anaerobic bacterias or just a
mixture of aerobes and anaerobes in the cul-de-sac only
[5][6]. This bacterial agent can destroy cilia of adjacent cells
in the tubal epithelium by secreting a toxin. The symptomatic
gonococcal PID usually begins during or just after the
menstruations. It is supposed that the gonococcus starts theinfection and, if the microorganism is not properly eliminated,
determines a mixed aerobic-anaerobic infection which
involves bacterias originating from vagina and cervix [11]. By
alterating the epithelium, N.gonorrhoeae creates a favorable
environment for progression of the aerobic-anaerobic
bacterias. Soper et al. [12] conducted a study in which theprimary and the reccurent PID were both associated withgonococcal infection. Nevertheless, the majority of pacientswith PID history did not present with gonococci, but with

Fiziologia - Physiology • 2023.1(104)other aerobic and anaerobic pathogens. This is significantly
relevant to the upper genital tract infection with Haemophilus
influenzae (H. influenzae) which was associated with severe
tubal disease such as pyosalpinx formation in each of thecases, suggesting the ascension from the lower genital tract
where is usually found [12].
The microbiome, represented by the community ofsymbiotic, commensal and pathogenic microorganisms, hasbeen of a major importance in the study of the human bodyover the years. The “Human Microbiome Project” (HMP),
launched in 2007 in the United States, characterized thehuman microbiome from normal volunteers from minimum
four sites of the body, vaginal site constituting the mostimportant [13]. Many researches proved how a genital
microbial disequilibrium can lead to infertility onset [14]. Dueto the combination of cultivation-dependent andindependent molecular-based techniques, it wasdemonstrated that only certain species of Lactobacillus
dominate the healthy genital flora [13]. Thus, the mostfrequently detected species are Lactobacillus crispatus,
Lactobacillus iners, Lactobacillus jensenii and Lactobacillus
gasseri [14]. On one hand, these microorganisms play afundamental role in keeping the reproductive tract balanced,
specifically by producing hydrogen peroxide and lactic acid
which maintains a lower pH and hinders the growth of otherpathogens and on the other hand in the implantation andpregnancy outcomes [13]. Other numerous bacterias wereidentified in healthy or unhealthy microbiota, being relevant
to mention: Mycoplasma, Ureaplasma, Staphylococcus,
Streptococcus, Gardnerella, Bacteroides, Haemophilus etc.
[14]. The decrease of Lactobacilli and the excessive growthof aerobes and anaerobes alongside the local inflammatoryenvironment characterize a well-known polymicrobial
disease, bacterial vaginosis (BV). In the latter, the infecting
bacteria will more easily disseminate by ascending to theupper genital tract, leading to PID and finally to TFI [15]. In
addition to this, studies of pregnant women demonstrated ahigher frequency of chlamydial or gonococcal infection in
those pacients with a Lactobacillus-lacking vaginal
microbiota [16]. Weisenfeld et al. [16] enrolled in a study 255
females who reported sexual activity with men infected with
C. trachomatis or with N.gonorrhoeae. The results haveshown 13 pacients tested positive for chlamydia of 32
exposed and 42 of 75 for gonorrhea. More relevant than this,118 enrolled females with BV were more likely to present
chlamydial or gonococcal infection than did those without BV[16]. A strong evidence from a meta-analysis study
conducted by van Oostrum et al.[17] is supporting the roleof BV in the etiology of tubal infertility. An approximately
one in five infertile pacients has BV and one in three has analterated vaginal microbiome [17]. Other important findings
in a study by Morgan et al. [18] from an infertility clinic
showed a BV prevalence rate of 9% in women withspontaneous pregnancy during the infertility investigations,a 22% prevalence rate in those diagnosed with tubal
infertility and a rate of 33% in pacients with an abnormal flora.

13

Although these data suggest a feasible role of BV in the
etiology of infertility, more investigations are needed to lend
support to the results [18].
An acquired gonococcal, as well as chlamydial
cervicitis, salpingitis or the presence of bacterial vaginosis
contribute to developing a subclinical PID [19]. There arefindings indicating that in the case of acute PID with nosymptoms and signs, one in four women with gonorrhea orchlamydia, and one in seven women with BV had subclinical
PID. It is considered that the latter is similar to acute PID
regarding long-term reproductive sequelae, especially tubal
infertility. Patton et al proved morphologic changes andalterations in the mucosal cilia’s function of the fallopian
tubes in the patients with acute and silent PID, suggesting
the relationship between TFI and prior infection with C.
trachomatis or N.gonorrhoeae in women with no history ofacute PID [20]. Despite the fact that the incidence of PID has
decreased, it is of a great importance to mention that therates of tubal infertility have remained unaltered, suggesting
the existence of many patients underdiagnosed withinfertility resulted after a pelvic inflammation [19].
Since 1984, the role of genital mycoplasmas ininfertile females was intensively evaluated. Although somestudies suggested a lack, or if any, a minor association
between genital mycoplasmas, PID and infertility [21] [22],many demonstrated the opposite. The results of an old
serological investigation by Moller et al. [23] are solid withthe fact that PID caused by Mycoplasma hominis (M.
hominis) can lead to tubal damage and peritoneal adhesions.
The researchers found the presence of three times more
often antibodies against M. hominis in infertile women withevident tubal impairment, than in those with apparently
normal histerosalpingographies, and almost all of thepatients with adhesions had antibodies [23]. Thanks to the
new accurate diagnostic methods, in particular topolymerase chain reaction (PCR) tests, many other bacteria
were brought to light. Lancet et al. [24] analysed 115
symptomatic women, 50% (58) representing females
histologically confirmed with acute endometritis. Of all, 9%were positive for Mycoplasma genitalium (M. genitalium).
The microorganism was identified in both endometrial andcervical samples in 40% (4) women and in 30% (3) in each
endometrial and cervical samples alone, highlighting thelack of linking M. genitalium infection with age, marital
status, age at first sexual intercourse or the number ofsexual partners [24].
Ureaplasma urealyticum (U. urealyticum) is
another bacteria from Mycoplasmataceae family, same as
mycoplasmas, sometimes isolated from inflammatedfallopian tubes, but with a controversly role in PID andinfertility, because of its prevalence in the healthy lower
genital tract of fertile women [5]. Some results indicate thatU. urealyticum is strongly linked to infertility in women, butwith the mention that the main cause is represented by themale factor. Men with ureaplasma present lower sperm
motility, a high number of abnormal morphological forms and14

a poor sperm quality [25].
Another contributing factor to the genital
inflammation and female infertility is represented bystaphylococcal infection. This aerobic /facultative anaerobic
cocci can cause inflammation by releasing cytokines andchemokines, resulting in menstrual and ovulatory
dysfunctions and failure of the fetal implantation.
Furthermore, the infiltration of plasma cells in the stroma of
the endometrium is usually a characteristic of the later stage,causing uterine bleeding and infertility [26]. A study from2014 [27] has identified Staphylococcus aureus (S. aureus)
as the most prevalent vaginal microorganism, with a 57.33%
percentage [27]. In contrast with this, in another study by
Cicinelli et al. [28] of 438 women with chronic endometritis
at hysteroscopy, S. aureus was present in 0 cases of thevaginal cultures and in 20 in those from the endometrium[28].
Female genital tuberculosis (GTB) is of a great
importance when referring to menstrual dysfunction andinfertility in developing countries. GTB is commonly present
as a secondary infection acquired from the lungs or from theabdomen by a hematogenous spread [29]. A recent study
showed that GTB is more frequent in infertile women even
where the prevalence of this disease in uncommon. Of a
total of 151 women (22.1%) with TFI, one third (31.1%) had
GTB [30]. The actual frequency of GTB is difficult to be
determined because of the poor clinical presentation whichleads to a late, or in some cases, omitted diagnosis [31].
Trichomoniasis, the most curable STD worldwide,is caused by the infection with an anaerobic protozoa,
Trichomonas vaginalis (T.vaginalis). Similar to chlamydia
and higher than gonorrhea, the prevalence of the affected
women in the USA is approximately 3.1%, with the mostnumerous cases in non-Hispanic black and post-adolescentfemales [32]. A percentage of 10.4% women werediagnosed with trichomoniasis after complaining of vaginal
discharge and other symptoms in the Republic of Korea [33].
Previously isolated from peritoneal liquids, nowadays more
attention is given to its possible role in the upper genital tract
infection and PID. Paisarntantiwong et al. has linked
trichomoniasis with PID in chlamydial-colonized women.
More than that, the results demonstrated an association
between T.vaginalis and the coinfection with Human
Immunodeficiency Virus-1 (HIV-1), with a higher risk of PID,
although the mechanism of this association is poorly
understood [34].
Other important findings show a potential
implication of Herpes Simplex virus-2 (HSV-2) in thefallopian tube obstruction and in acute endometritis,
facilitating the development of PID, but more research isneeded to support these results [35].
CONCLUSIONS
The most optimum way to prevent the genital tract
infections, PID and the consecutive sequelae (e.g. ectopic
pregnancy, infertility etc.) consists of thorough screening,

Fiziologia - Physiology • 2023.1(104)early and, when necessary, aggressive treatment [36].
Coupled with all these, the application of newtechnological advances in specimen collection and in theidentification of STDs offers a great expectation in reducing
the incidence of infertility due to infections [5].

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INFECȚIILE GENITALE: O CAUZĂ MONDIALĂ A INFERTILITĂȚII FEMININEREZUMAT
Infertilitatea reprezintă în majoritatea cazurilor o tulburare multifactorială, definită ca incapacitatea de a concepe după un an de contact sexual,în lipsa oricărei metode contraceptive. Există o multitudine de cauze dovedite care afectează fertilitatea feminină, pe langă vârsta avansată:patologiile uterine, disfuncțiile tubare și ovulatorii. Afecțiunile tubo-peritoneale reprezintă grupul cel mai important care determină apariția bolii
inflamatorii pelvine (BIP) și a infertilității. Infecțiile genitale netratate, ca de exemplu clamidiaza, gonoreea și alte infecții cu microorganisme
aerobe sau anaerobe sunt semnificativ relevante pentru distrucțiile uterine și tubare, determinând în cele din urmă infertilitate feminină.
Microbiomul uman joacă un rol esențial în menținerea tractului reproductiv echilibrat, în special datorită prezenței anumitor specii de lactobacili.
Studiile au demonstrat că o scădere a numărului de lactobacili și o creștere excesivă a altor patogeni, alături de inflamația local, duc la apariția
unei boli bine cunoscute, vaginoza bacteriană (VB), de asemenea implicată în infertilitate. Noile progrese tehnologice și screening-ul intensiv
constituie factorii cei mai importanți în reducerea incidenței infecțiilor genitale și oferă rezultate pozitive în evolutia sarcinii.
Cuvinte cheie: infertilitate, boală inflamatorie pelvină (BIP), infertilitate tubară, microbiom

Titlul proiectului: ROHU-339, „Romanian-Hungarian cross-border project with a wider focus on diagnostics related toinfertility, healthy pregnancy and newborn care”, acronim HEALTH-PREGN-RO-HU
Editorul materialului: Spitalul Clinic Județean de Urgență „Pius Brînzeu” Timișoara
Data publicării: aprilie 2023
Acest proiect este cofinanțat de Uniunea Europeană prin Fondul European de Dezvoltare Regională în cadrul Programului Interreg VA România-Ungaria.
Conţinutul acestui material nu reprezintă în mod necesar poziţia oficială a Uniunii Europene.

16

Fiziologia - Physiology • 2023.1(104)ALTERATION
OF
SOME
RED
BLOOD
COMPONENTS IN DIABETES MELLITUS

CELL

MATEI DANIELA MARIANA1
University of Oradea, Faculty of Medicine and Pharmacy Oradea, Department of Preclinical Disciplines, dmcernau@gmail.com

ABSTRACT
Diabetes mellitus (DM) is a metabolic disease associated with many complications. The hyperglycemic and dyslipemic plasma environment fromDM determines qualitative and quantitative changes in the (RBC) membrane, enzymes, transporters and hemoglobin. Hyperglycemia can
influence the osmotic resistance of RBCs, the activity of transporters and pumps in the cell membrane, the content of 2,3-diphosphoglycerate(2,3-DPG), the secondary structure of hemoglobin and its affinity for oxygen, the intracellular redox balance, and eventually, the delivery of oxygento the cells of the body. The biochemical, structural, and functional abnormalities of RBCs observed in DM appear to be related to the glycemic
status of patients. At least at the level of RBCs, several "targets" could be affected by elevated blood glucose, even though this paper reviewed
only a few of them. Knowing the consequences of hyperglycemia and dyslipidemia on the morphology and functional characteristics of RBCs
can be useful in understanding the effects of these factors on other cells of the body and finding effective therapies to prevent this disease andits complications.
Keywords: diabetes mellitus, protein glycation, RBC metabolism, transporters, 2,3-DPG

INTRODUCTION
Diabetes mellitus (DM) is a chronic metabolic
disease characterized by disturbances in carbohydrate, lipid
and protein metabolism as a result of reduced insulin
secretion and/or insulin resistance of peripheral tissues.
Insulin deficiency causes hyperglycemia, abnormal lipid
profile and reduced protein synthesis. When red blood cells(RBCs) are chronically bathed in plasma of abnormalcomposition, biochemical changes occur in the RBC
membrane, enzymes, transporters, and hemoglobin, which
will impact the normal metabolism and redox status of thesecells and ultimately the ability to perform their normal
functions. This article is a brief review of some RBC
alterations associated with hyperglycemia and dyslipidemia
in DM.
The particular geometry of RBC gives it the abilityof resisting distortion without membrane rupture andrestoring its shape after repeated passage through bloodvessels during the 120 days it remains in circulation [1-3].
However, physiologically, as RBCs age, deformability [1],surface area and zeta potential decrease [4], denatured
hemoglobin accumulates, and cells become more rigid andsmaller [4]. These changes make RBCs less adapted to theosmotic and mechanical stressors they are subjected to in
the circulation.
The major factors affecting RBC deformability arecell geometry (size and shape), cytoplasmic viscosity, andviscoelastic properties of its membrane [1, 5]. All these threecomponents could be altered in DM. The membrane islargely responsible for the biconcave shape, pliability andresiliency of RBCs [6]. The RBC membrane resists bending
but cannot sustain in-plane static shear stress [3].

Cytoplasm viscosity depends on hemoglobin concentrationand solubility [1, 5, 7], whereas cell membrane viscosity on
RBC membrane components [5] and their structure.
ALTERATIONS OF THE PROTEINS AND LIPIDS IN THE
RBC MEMBRANE
One of the consequences of hyperglycemia is
glycation (glycosylation) of hemoglobin and RBC membraneproteins [7-9]. Protein glycosylation, in turn, can lead to RBC
membrane abnormalities observed in DM [8, 10].
Glycosylation of hemoglobin can change its affinity foroxygen [11]. In a study on diabetic RBCs by Schwartz et al.
[10], it was showed that several RBC membrane proteins
were heavily glycosylated compared to nondiabetic RBCs
and among these proteins, β-spectrin, ankyrin, and protein
4.2 were the most glycosylated [10]. In addition, diabetic
spectrin was biochemically altered, containing significantly
less methionine and histidine, but a double amount of cysteic
acid compared to normal spectrin [10]. The deformability of
diabetic RBC as measured by ektacytometry was lower thanin control subjects, and diabetics with low RBC deformability
had a higher level of glycosylated hemoglobin than diabetics
with normal RBC deformability [10]. They concluded thatspectrin glycosylation induces its oxidative damage that
decreases RBC deformability [10].
Nonenzymatic glycosylation of normal, human
RBC proteins has also been observed in vitro studies inwhich normal RBCs were incubated with various
concentrations of glucose [8, 12]. This caused levels ofprotein-bound glucose and anisotropy values similar to
those found in patients with DM [8]. Moreover, a linear
correlation was found between the amount of lysine-bound17

Fiziologia - Physiology • 2023.1(104)glucose of total hemoglobin and membrane proteins and
RBC membrane fluidity [8]. The fluidity of RBC membraneghosts was also assessed in diabetic juvenile patients bymeasuring membrane anisotropy [9]. A significant increasein anisotropy was found in diabetic RBC membranes
compared to controls [9], associated with an increase innonenzymatic glycosylation of membrane proteins andhemoglobin [9]. A linear relationship was observed between
glycosylated hemoglobin and glycosylated membraneproteins, on the one hand, and glycosylated hemoglobin andmembrane anisotropy, on the other hand [9]. Watala et al. [9]suggested that the enhanced glycosylation of membraneproteins that occurs in diabetes could be one of the reasonsfor the decreased membrane fluidity of diabetic RBC
membrane [9].
A parameter that appears to be altered by theglycation level of hemoglobin in patients with DM is the
dynamic fluctuations of RBC cell membrane thickness [13].
Sohn et al. [13] studied the deformability of RBCs frompatients with DM by evaluating the dynamic fluctuations of
RBC cell membrane thickness using the holotomography
technique [13]. They reported a negative correlation
between dynamic fluctuations in RBC cell membranethickness and glycosylated hemoglobin A1c (HbA1c)
concentration and fasting plasma glucose level [13]. Whenthe dynamic fluctuations in RBC membrane thickness frompatients with DM were compared with the control group, it
was significantly lower [13]. This parameter was alsosignificantly lower in patients with cardiovascular diseases,
diabetic nephropathy, diabetic retinopathy, hypertension anddyslipidemia compared with those without these conditions[13]. In addition to dynamic fluctuations in RBCs membranethickness, researchers analyzed other morphologicalparameters such as cell volume, RBC surface area,
sphericity index, and hemoglobin protein density [13]. They
reported significantly greater RBC volume and surface area
in DM patients compared to healthy subjects [13].
Analyzing the shape of diabetic RBCs under aphase contrast microscope, abnormal configurations ofRBCs
associatedwithhyperglycemia
andhypercholesterolemia were observed, and these changes
were more significant when hyperglycemia was associatedwith hypercholesterolemia than with normal blood
cholesterol levels [14]. Compared to normal RBCs, thosefrom diabetics showed an increased perimeter and reduced
surface area, a significantly increased perimeter/area ratio,
and an increased form factor [14]. The form factor is anindicator of the roundness of RBCs, and its variation
represents a deviation from the normal biconcave shape [14].
The significant increase in form factor in RBCs from patientswith hyperglycemia and hypercholesterolemia shows that
RBC shape has deviated from normal, and this change maylead to decreased deformability and increased aggregabilityof RBCs [14]. Indeed, analyzying the parameters of RBC
aggregation, Babu [15] reported that they were decreased indiabetic patients with normal cholesterolemia and18

significantly
decreasedindiabetics
withhypercholesterolemia compared to the control group,
denoting that RBC aggregation was significantly increasedin patients with DM and hypercholesterolemia, and therefore
a higher risk of vascular complications for these patients [15].
Increased RBC membrane cholesterol content due tohypercholesterolemia appears to impair RBC deformability,
resulting in decreased membrane fluidity [2]. The effect ofhypercholesterolemia in reducing RBC deformability was
also demonstrated by another study [16].
RBC membrane is composed of 52% proteins, 40%
lipids (phospholipids, cholesterol, triglycerides) and 8%
carbohydrates [15]. About half of the phospholipid bilayer ismade up of cholesterol. The exchange between plasma andRBC membrane cholesterol can alter the cholesterol content
of the cell membrane [17]. Cholesterol accumulation in the
RBC membrane results in abnormal RBC morphology [15],can shorten RBC survival [6], and causes a decrease in RBC
deformability [15, 17]. The extent of the shape changes
depends on the amount of cholesterol incorporated into themembrane [18]. To assess the influence of lipids on the RBC
membrane, Cooper et al. [17] subjected RBCs tocholesterol-lecithin dispersions with variable amounts ofunesterified cholesterol relative to phospholipid (lecithin)[17]. Changes in membrane cholesterol content were
followed by changes in membrane surface area [17].
Cholesterol-poor RBCs became spherocytic in appearance,
while cholesterol-rich RBCs were broad and flat with anirregular contour [17]. The addition of phospholipid
prevented the increase in membrane cholesterol andsurface area and the change in cell shape [17]. Cholesterol
makes the RBC`s membrane less rigid and more resistantto the diffusion of water inside of RBCs when they are
immersed in an hypotonic medium [15, 17]. A lower blood
cholesterol level depletes the RBC membrane of cholesterol
and makes the cells osmotically fragile, whereas RBCs thatgain cholesterol are resistant to osmotic lysis [17]. Several
studies have shown that long-term administration ofcholesterol-lowering therapy (hepatic hydroxymethyl glutaryl
coenzyme A reductase inhibitors) in patients withhypercholesterolemia
reducedbloodcholesterol
concentration and improved RBC deformability [2].
ALTERATION IN THE ELECTRICAL CHARGE OF RBCMEMBRANE SURFACEThe effects of hyperglycemia are not limited tochanges in membrane proteins and lipids or the microscopic
appearance of RBCs, but also to the reduction of negative
charges on the RBC membrane [15] that are largely due tothe presence of the carboxyl group of sialic acids [19] of theouter surface of the cell membrane. The normal negativeelectrical charge of RBC surface membrane (zeta potential)
causes RBCs to repel each other and to be repelled by othercells [19], and also by the endothelial glycocalyx [20].
However, there is no unanimous opinion that theglycosylation of RBC membrane proteins reduces the

Fiziologia - Physiology • 2023.1(104)electrical charge of the membrane surface [21], even thoughmany studies have reported a decrease in the negative
charge of the RBC membrane in DM [21-23]. In a researchon diabetic RBCs regarding their electrical behavior
analyzed by dielectric spectroscopy [21], the relative
permittivity, dielectrical loss, and electrical conductivity (ACconductivity) of diabetic RBCs were significantly increased
compared to control RBCs [21]. This could be the result of
toxic effect exerted by glucose on RBCs, which leads to therestructuring of the RBC membrane [21]. RBC membrane
glycosylation reduces the negative electrical surface charge
due to cleavage of the terminal sialic acid components ofglycophorin A, leading to accelerated RBC aging [21].
BudaK et al. [22] reported that RBC anionic charge wassignificantly decreased in diabetic patients with retinopathy
compared to control subjects [22, 24] and decreases as thegrade of diabetic retinopathy increases [24]. A statisticallysignificant reduction in RBC anion charge and sialic acid andacid glycosaminoglycan content in phantom RBCs frompatients with insulin-dependent DM and microalbuminuria
compared with control subjects has been reported [25].
Bernard et al. [23], using the Alcian Blue binding assay,
found that RBC membrane negative charges were
diminished in patients with DM and were negatively
correlated with urinary excretion of albumin and other highmolecular weight urinary proteins [23]. The decrease innegative RBC surface potential enables macrophages toovercome the electrostatic barrier, to get closer to RBCs andphagocytose them [4].
ALTERATION OF OSMOTIC AND MECHANICALRESISTANCE OF RBC
There is increasing evidence that anemia is one of
the common complications in patients with type 2 DM [26,27]. Even though the causes and pathogenetic mechanismsof this condition in DM patients are not fully settled, being
complex and multifactorial [27], potential explanations,among many others, could be RBC membrane alteration[26], increased osmotic [24, 26] and mechanical fragility of
RBCs [27] due to the structural changes of the membrane
induced by the hyperglycemic and dyslipemic environment,
which accelerates their destruction in the spleen [27].
Diabetic RBCs have a significant increased mechanicalfragility than normal RBC, and furthermore, it was positively
correlated with fasting blood glucose and glycosylated
hemoglobin A1c (HbA1c) [27]. Regarding the osmotic
fragility of diabetic RBCs, it has been reported to be higher
in diabetics compared to healthy subjects [26, 28, 29] andcorrelated with duration of DM [26]. In another study [30],increased osmotic fragility of diabetic RBCs was significantly
correlated with fasting blood glucose and two hours post
prandial blood sugar [30]. Mechanisms underlying increased
osmotic fragility may be membrane protein glycosylation andalteration of RBC membrane Na+/K+-ATP-ase [2, 27], butalso the impact of abnormal cholesterolemia. The osmotic
fragility test of diabetic RBCs showed that these cells weremore susceptible to hemolysis than those from healthy
volunteers after 3 hours of incubation with 0.7% v/v NaClsolution, probably due to glycated hemoglobin [31]. RBC
osmotic fragility was positively correlated with glycosylated
hemoglobin [28].
A potential factor associated with increased RBC
osmotic fragility in DM is RBC ascorbate deficiency [32]. The
dominant human RBC transporter for glucose is GLUT1,
which is also used for the transport of dehydroascorbic acic[32]. In vitro studies [32], showed that glucose anddehydroascorbic acid appear to have similar affinities forfacilitated transporters [32]. Experimental observations inmice and humans indicated that RBC ascorbate
concentrations were inversely related to hyperglycemia [32]and likely that dehydroascorbic acid competes with glucose
for being transpostered into RBC. Low RBC ascorbate could
contribute to anemia by maintaining a low level of chronic
hemolysis by increasing the RBC osmotic fragility [32].
Human RBC ascorbate concentrations were inversely
related to glycemia, osmotic fragility, and severity of DM [32].
ALTERATION OF RBC Na+-K+-ATP-ase ACTIVITY
The energy requirements of RBCs are provided by
glucose metabolism. Glucose is needed to maintainhemoglobin in a soluble, reduced state, to provide 2,3
dyphosphoglycerate (2,3-DPG), and to generate ATP to
support membrane stability and maintain cell shape anddeformability [6]. ATP depletion of RBC is associated withdiscocyte-echinocyte transformation and accumulation of
calcium [33]. RBC Na+-K+-ATP-ase has a key role inmaintaining the cation gradient across the cell membrane[34, 35] and cell volume and thereby cell deformability [2].
Impairment of Na+-K+-ATP-ase activity results in intracellularsodium accumulation and increased extracellular potassium
concentration. Insulin increases Na+-K+-ATP-ase activity[36]. Insulin stimulates Na+/H+ exchange transport, and theresulting Na+ influx increases the intracellular Na+
concentration, thus activating Na+/K+-ATP-ase [36]. In
mammalian cells, insulin can also promote transcription andtranslation of Na+-K+-ATP-ase or reduce degradation of the
α1 subunit of the pump [35].
Altered activity of this pump is thought to berelated to several complications of DM [34]. RBC Na+-K+ATP-ase activity was found to be significantly reduced indiabetic patients compared to the controls [34], and the
enzyme level was negatively correlated with DM duration[34]. The enzyme activity was significantly lower in patientswith diabetic neuropathy [34], and also in those with diabetic
retinopathy compared to those without this complication [34],but the pump activity did not differ among the degrees ofdiabetic nephropathy [34]. Minura et al. [37] investigated
RBC Na+-K+-ATP-ase activity in diabetic patients with andwithout microalbuminuria and showed that enzyme activity
was significantly reduced in the diabetic group withmicroalbuminuria
comparedtothose
without
microalbuminuria and control subjects [37]. Raccah et al.[38]19

Fiziologia - Physiology • 2023.1(104)reported a significant lower Na+-K+-ATP-ase activity in agroup of patients with long-lasting insulin-dependent DM
compared to control group, but its activity was not dependent
on the degree of diabetic control, nor was correlated witheither fasting blood glucose or HbA1c [38]. It was lower indiabetic patients with neuropathy compared with those
without it [38] and positively correlated with nerve
conduction velocity in the peroneal and tibial nerve [38].
However, a previous study [29] found an increase RBC Na+K+-ATP-ase activity in diabetic patients [29]. In vivo insulin
treatment restored the enzyme activity and the osmotic
fragility of diabetic RBCs to the normal level [29]. In vitro
insulin treatment of diabetic RBCs inhibited the further
increase in its activity, but could not restore the activity to thenormal level as in vivo [29].
ALTERATION OF RBC Ca2+-ATPase ACTIVITY AND
BAND 3 PROTEIN
RBC deformability and elasticity depend on theintracellular concentration of Ca2+ ions, the level of which isregulated by the plasma membrane Ca2+-ATPase [39]. The
pump removes Ca2+ ions from RBCs. Alteration of its activity
is followed by Ca2+ accumulation that can increase RBC
membrane rigidity [33, 40], change the shape and volume,and eventually induce hemolysis [33]. ATP, Mg2+ and Ca2+
control the shape of RBC through their interaction withinternally located fibrillar proteins, which are involved inmaintenance of the disc shape of RBC [33]. When basal andcalmodulin-activated ATP-ase activity was compared
between diabetics and healthy subjects, a significant
reduction in basal and stimulated activity of the enzyme wasreported in diabetic subjects compared to healthy
participants [40]. The reduction of Ca2+-ATP-ase activity wasobjectified in vitro experiments, in which RBCs were
exposed to normal (5 mM) and high concentrations of
glucose for variable time intervals [39]. Among many otherparameters assessed in this research, the investigators
reported calcium accumulation in RBCs. Intracellular
calcium concentration in both control and high glucoseexposed RBCs increased after 48 and 72 hours andcorrelated with decreased plasma membrane Ca2+-ATP-ase
activity [39]. Experimentally, an increase in RBC
concentration of calcium leads to changes in cell size
(shrinkage), shape (appearance of spherocytes andechinocytes) and cation transport across the RBC
membrane [41], and ultimately causes hemolysis [39].
Inactivation of the Ca2+ pump is a consequence of glycationof this protein [42].
A significant decrease in RBC membrane Ca2+ATPase activity in non-insulin-dependent DM patientscompared to healthy subjects was reported by Ramana et al.
[43]. Researchers also found a significant reduction inmembrane glycoprotein in DM patients and a significant
increase in erythrocyte total Ca2+ compared to controls [43].
Decreased Ca2+-ATPase activity would lead to increased
erythrocyte total Ca2+ levels as a result of permeability

20

alterations [43], and its accumulation leads to the loss ofmembrane integrity [43].
Another protein, whose expression levelmeasured in the RBC was significantly lower in diabetic
volunteers compared to healthy ones, was Band 3 protein[31]. In DM, decrease in the Band 3 protein level is due tohemoglobin glycation and is increased by oxidative stress[31]. Band 3 protein is a Cl-/HCO3- exchanger. Anion
exchange is carried out by the multi-spanning integral
membrane domain of protein [44]. The functional role of thisexchanger in facilitating the transport of carbon dioxide iswell known. In addition, this protein has an important role inmaintaining normal biconcave RBC shape, membranestability, and resilience to share force [44]. The Band 3
protein is bound to spectrin via ankyrin, showing that it isinvolved in membrane-cytoskelton interaction to maintain
RBC shape and stability [44]. Animal studies have shownthat Band 3 protein deficiency leads to chronic hemolytic
anemia [44]. Its deficiency results in loss of RBC surface
area and a tendency to acquire the round shape (spherocyte)[44].
ALTERATION IN THE SECUNDARY STRUCTURE OFHEMOGLOBIN
Hemoglobin is the main protein of RBC cytoplasm,
and changes in its structure influence RBC function [7, 45].
Throughout the lifetime of RBCs in circulation, glucose
nonenzymatically attaches to the N-terminal amino group of
the beta chain of hemoglobin by a ketoamine likage forming
glycosylated (glycated) hemoglobin A1c (HbA1c) [46, 47].
The initial reversible condensation of glucose andhemoglobin forms an aldimine or Schiff base, also known aslabile HbA1c, which undergoes an irreversible
intermolecular Amadori rearrangement to form ketoamine,
known as stable HbA1c [47]. The α-amino group of the beta
chain is involved in the normal function of hemoglobin [47].
This group lies in the central cleft of the molecule, being
directly involved in the binding of glucose, 2,3-DPG, carbon
dioxide and hydrogen ions [47]. Binding of oxygen tohemoglobin alters the position of this group [47]. The HbA1cvalue increases with plasma hyperglycemia [7, 45] andcorrelates with the average glycemia over the last two or
three months [46]. Many other proteins, including RBC
membrane proteins, are modified by nonenzymatic
glycosylation [46]. Glycosylation-induced structural
alteration can lead to impairment of protein functions [46].
Structural changes induced by hemoglobin glycation may
lead to oxidative stress in DM [45]. The risk of diabetic
complications is related to the level of HbA1c [7, 46].
The effect of glucose on hemoglobin secondary
structure was analyzed by Fourier transform infrared
spectroscopy [7]. Ye et al. [7] found that the secondary
structure of hemoglobin had a strong tendency to changewith increasing glucose concentration levels, andhyperglycemia can affect both RBCs and hemoglobin [7].
The secondary structure of a protein influences the

Fiziologia - Physiology • 2023.1(104)conformation of that protein [7]. The proportion between αhelix and β-sheet content in the protein structure changes
the solubility of that protein [7]. The insolubility of proteins
increases proportionally with the content of the β-sheet
structure, causing their aggregation [48]. In the study
conducted by Ye et al.[7], diabetic patients with goodglycemic control (HbA1c < 7%) showed a slight change inthe content of α-helix and β-sheet structures compared tothe control group, whereas those with a poor glycemic
control (HbA1c ≥ 9%) presented a significant increased inthe β-sheet content of hemoglobin compared to the control
group [7]. In addition, compared to diabetic patients in thegroup with good glycemic control, those with poor glycemic
control showed a significant increase in the content of βsheet structures and a decrease in α-helix structures as the
HbA1c concentration increased over 9% [7]. Prolonged
exposure of RBCs to hyperglycemia induces chemical
modifications in the structure of hemoglobin that decrease
structural stability, causing its aggregation [7]. Decreased
hemoglobin solubility increases RBC cytoplasmic viscosity[7], reduces RBC flexibility, and negatively impacts RBC
circulation through capillaries, predisposing diabetics tovascular complications [49]. Similar observations regarding
hemoglobin secondary structure caused by RBC exposure
to abnormal glucose concentrations were made by Andleeb
et al. [50]. The Fourier transform infrared spectroscopy
technique used to analyze the hemoglobin structure showedthat the hemoglobin structure was slightly altered in thegroup of diabetics with good glycemic control (6% < HbA1c< 7%), but significantly altered in the group with poor
glycemic control (HbA1c ≥9%) [50]. There was an increasein β-sheet structure and a decrease in α-helix structure athigh HbA1c levels (diabetics with HbA1c ≥9%) [50].
ALTERATION
OF
2,3-DIPHOSPHOGLYCERATECONCENTRATION
From a functional point of view, RBC hemoglobin
has the role of transporting oxygen and carbon dioxide andbuffering the hydrogen ions released by dissociation of acids.
The amount of oxygen transported to the tissues depends
on the blood concentration of hemoglobin and its affinity foroxygen. The affinity of hemoglobin for oxygen is modulated
by several factors. The ease with which hemoglobin unloads
oxygen to tissues is influenced by 2,3-DPG [51]. An increasein the concentration of 2,3-DPG decreases hemoglobin
affinity for oxygen and improves tissue oxygenation, while adecrease in its concentration will result in the opposite effect,
predisposing cells to hypoxia [51]. 2,3-DPG is synthesizedin RBC from glucose. The unique source of energy for RBCs
is the anaerobic metabolism of glucose via glycolysis tolactate [52]. Insulin increases the rate of glycolysis in human
RBCs [52]. The synthesis and breakdown of 2,3-DPG iscontrolled by Rapaport-Luebering shunt, a side-pathway of
Embden-Meyerhof pathway [51].
Data on 2,3DPG concentration in DM patients areconflicting in the literature. Previously, some researchersreported that insulin administration was associated with adecrease in 2,3-DPG concentration [53, 54], but otherstudies reported a significantly elevated level of 2,3-DPG inpatients with insulin-dependent DM compared tonondiabetic subjects and a more reduced increase in noninsulin-dependent DM patients [55]. However, the overall
position of the oxyhemoglobin dissociation curve was thesame for diabetic patients and nondiabetic subjects, despiteof the fact that increased glycosylated hemoglobin indiabetic patients was expected to shift the oxyhemoglobin
dissociation curve to the left [55]. In patients with insulindependent DM, increased 2,3-DPG appeared tocompensate for the effect of the glycosylated hemoglobin on
the position of the oxyhemoglobin dissociation curve [55]. It
is known that 2,3-DPG is not the only factor changing theaffinity of hemoglobin to oxygen. Acidosis and increased
partial pressure of carbon dioxide decrease hemoglobin
affinity for oxygen as well.
A slight elevation in 2,3-DPG was reported in agroup of patients with type 2 DM and a HbA1c concentration
above 7% compared to a group of type 2 diabetic patientswith HbA1c ≤ 7% [56]. The arterial oxyhemoglobin
dissociation curve for patients with HbA1c > 7% shifted to
the left compared to the curve for those with HbA1c ≤ 7%[56]. Saturation of arterial blood with oxygen (SaO2)(determined for identical partial pressure of oxygen) was
higher in type 2 diabetic patients with HbA1c > 7% than indiabetic patients with HbA1c ≤ 7%, which is likely due toincreased affinity of HbA1c for oxygen [56]. Also, pulse
oximeter oxygen saturation (SpO2) was higher in diabetics
with poor glycemic control (HbA1c >7%) than in those with
better glycemic control (HbA1c ≤ 7%). An important findingof the study [56] was that in patients with type 2 DM withpoor glycemic control, pulse oximetry overestimated arterial
blood saturation compared to patients with better control inwhom mean SpO2 values were approximately the same [56].
In contrast, Triolo et al. [57] showed that the percentage ofglycosylated hemoglobin was significantly inversely related
to the RBC concentration of 2,3-DPG [57]. The more poorly
controlled the diabetes, the lower the RBC 2,3-DPG
concentration value [57]. In another study [58], even though
the measured concentration of 2,3-DPG in diabetic RBCs
was significantly higher than in control subjects, it did notcorrelate significantly with glycemic equilibrium [58].
Abnormal 2,3-DPG levels in diabetics appear to be related
to the presence of vascular complications of DM and not tothe duration of DM [59]. The concentration of 2,3-DPG wasfound to be normal in diabetics with no evidence of vascularcomplications, but significantly higher in those with vascularcomplications compared to healthy subjects and diabetic
patients without vascular complications [59]. The increased
concentration of 2,3-DPG may be the result of RBC
attempting to compensate for tissue hypoxia in diabetics
with vascular disease [59].
HbA1c has a higher affinity for oxygen [11, 58],
approximately ten times higher than non-glycosylated
21

Fiziologia - Physiology • 2023.1(104)hemoglobin [11]. Glycation of hemoglobin increases
hemoglobin-oxygen affinity and reduces delivery of oxygento tissues by altering the structure and function ofhemoglobin [56]. Nervertheless, the oxygen affinity in bloodfrom diabetic patients does not differ from that in healthy
subjects [11]. According to Marschner et al. [11], 2,3-DPG
which was increased in diabetic patients [11] compensatedfor the increase in HbA1c affinity [11]. The oxygen
dissociation rate constant of hemoglobin was found to notdiffer between diabetic patients and healthy subjects, hence
it appears that 2,3-DPG may not be the major factor
compensating for increased HbA1c affinity and oxygen
release in diabetic patients [11]. A research done by Bard etal. [60] on newborn lambs that were perfused several days
with a hypertonic glucose solution showed a significant
decrease in the concentration of 2,3-DPG in RBCs fromlambs subjected to hyperglycemia compared to the control
group of newborn lambs perfused with physiological saline[60]. The decrease in 2,3-DPG concentration can be
explained by the metabolic changes within RBC. Exogenous
glucose could suppress the 2,3-DPG synthesis by inhibiting
glucose phosphorylation as well as increasing the activity ofthe polyol pathway [60], producing sorbitol [61].
ALTERATIONS OF RBC METABOLISM
Glucose enters RBCs by facilitated diffusion,
which is an insulin-independent process [61]. The uptake isdone by glucose transporter 1 (GLUT1), an integral
membrane hydrophobic protein, having specificity for
glucose and galactose [62]. This is the main glucose
transporter of the RBC [63]. In human RBC, GLUT1
comprises about 5 -10% of the total membrane protein[62]. GLUT1 expression is mainly regulated by glycemia, cellsignaling mechanisms and hormones [62]. In hypoglycemia,
there is an upregulation of GLUT1 in tissues [62].
Inside the RBC, the phosphorylation of glucose to
glucose-6-phospate is catalyzed by hexokinase. RBC
hexokinase has a very low Km (Michaelis constant – ameasure of the affinity of an enzyme for its substrate) for
glucose and is saturated even at normoglycemic levels [64],while the Km of RBC aldose reductase (the enzyme thatcatalyzes the reduction of glucose to sorbitol) is much higher[61, 65]. Therefore, when glucose supply to RBCs is
increased, as in hyperglycemia, more glucose could be
metabolized via the polyol pathway [64, 65] and only a small
percentange via the glycolytic pathway [64]. The RBC
membrane is not permeable to sorbitol, but it can bedegraded by sorbitol dehydrogenase and converted tofructose [61, 65]. The reduction of glucose to sorbitol byaldose reductase consumes NADPH, whereas theconversion of sorbitol to fructose by sorbitol dehydrogenase
is at the expense of NAD+, leading to the production of
NADH [65]. The higher the glucose level, the higher the
NADH concentration [65]. Accumulation of sorbitol, fructose
and NADH and consumption of NADPH have been
implicated in pathogenesis of DM and its complications [65].

22

RBC concentration of sorbitol was found in higher
concentrations in type 1 (insulin-dependent) DM patientscompared to healthy subjects [66]. In vitro experiments, inwhich RBCs from type 1 DM patients were exposed tonormal extracellular glucose concentrations for 3 h, they
accumulated more sorbitol and fructose than normal RBC
under the same experimental conditions [66]. Nearnormalization of glycemia in type 1 diabetics for 12 hours did
not bring back to normal RBC sorbitol level [66].
Aldose reductase is stimulated by glucose-6phosphate and inhibited by 2,3-DPG [61]. Under
hyperglycemic conditions, more glucose-6-phosphate andless 2,3-DPG could be produced [64]. Therefore, sorbitol
synthesis may be enhanced at higher blood glucose levels[61]. Incubation studies on RBCs from healthy and diabetic
subjects indicated that RBC sorbitol concentration oscillated
in parallel with the concentration of ambient glucose [61].
RBC sorbitol increases rapidly when extracellular glucose
concentration is elevated and decreases when glucose
levels decrease [61]. However, in vivo studies [66], reported
that in a hyperglycemic milieu, sorbitol concentration in
RBCs increased but it did not decrease when glycemia islower [66]. Intracellular accumulation in hyperglycemic
conditions of glucose and sorbitol increases cellularosmolarity and influences the size and shape of RBCs, butalso through complex chemical reactions the normal
functioning of RBCs. Defects in anaerobic glycolysis areassociated with increased cell rigidity and decreased RBC
survival, and may therefore cause hemolytic anemia [6].
Synthesis of ATP and 2,3-DPG requires
phosphate [67]. Decreased intracellular phosphate occurs indiabetic ketoacidosis, and the cause of this is in part the shiftof phosphorus from the intracellular to the extracellularcompartment and increased urinary phosphorus excretion[67]. Low level of intraerythrocytic phosphate inhibits
glycolysis whereas high levels stimulate it [68]. In acidosis,
the oxyhemoglobin dissociation curve initially shifts to theright, but this will be counterbalanced over the next few
hours by a compensatory decrease in RBC 2,3-DPG levels
and the curve returns to normal [6].
The increased activity of the polyol pathway in DM
interferes with the redox state of RBCs, reducing theconcentration of NADPH and NAD+ [65]. Decreased NADPH
concentration can induce oxidative stress by altering
glutathione metabolism [65, 69]. Depletion of glutathioneleads to the inability of RBCs to counteract the effect ofoxidative stress on hemoglobin and RBC components [69].
Denaturated, oxidized hemoglobin precipitates on the innersurface of the RBC membrane [6], causing membrane
damage and hemolysis by macropahges of thereticuloendothelial system [6, 69]. This may also contributeto anemia of DM [69].
The end product of glucose fermentation is lactate.
RBC carbonic anhydrase activity and blood lactate
concentration were found to be significantly higher inpatients with type 2 DM compared to the healthy subjects

Fiziologia - Physiology • 2023.1(104)[70]. The increase activity of carbonic anhydrase could be
the result of increased glycolytic pathway that generates
more lactic acid and implicitly lactate [70]. Lactate cannot be
further metabolized by RBCs, so it accumulates if it is notremoved from RBCs. The efflux of lactate is mediate bymonocarboxylate transporters (MCTs) in an electroneutralmode of 1H+:1lactate [71]. Proton-coupled monocarboxylatetransporters are carriers for lactate, pyruvate, and ketone
bodies, being expressed in most tissues [71]. MCT1serves
both as a lactate importer and exporter, while MCT4 is themain exporter of lactate from cells producing large amountsof lactate [71]. The increase in carbonic anhydrase activity
could be done to facilitate lactate efflux from RBCs, toprevent its intracellular accumulation [70]. The blood lactate
is uptaken by liver and used as substrate forgluconeogenesis[70].
Therefore,
increasedgluconeogenesis in diabetics may be the consequence ofincreased carbonic anhydrase activity to stimulate lactate
efflux from RBCs [70]. Increased lactate and carbonic
anhydrase activity may contribute to decreased pH in RBCs,
which may interfere with increased glycation of hemoglobin[72]. The pH in RBC influences the rate of glycation ofhemoglobin [72]. Intracellular acidosis increases it, whilealkalosis decreases it [72]. Thus, hyperglycemia in DM
causes RBCs to enhance glycolysis, produce more lactatethat is followed by decrease of intracellular pH. In order toremove the excess of lactate from RBC, the activity ofcarbonic anhydrase should increase [70].
Human RBCs express insulin receptors andrespond to insulin by increasing tyrosine residue
phosphorylation in several proteins [52]. However, themetabolic effects associated with activation of this receptor
in RBCs are not clear [52]. Zancan et al. [52] showed that
RBC lactate production increased in the presence of insulin[52]. Insulin increases the rate of glycolysis in RBCs by
activating of a key enzyme, 6-phosphofructo-1-kinase [52].
Insulin increases the phosphorylation of this enzyme andredistributes the enzyme into the RBC, causing the enzyme
to detach from the RBC membrane [52]. Therefore, under
the action of insulin, the amount of enzyme associated withthe membrane decreases greatly [52]. Detachment is amechanism to activate the enzyme and prevent RBC
hemolysis [52]. Thus, in DM, due to insulin deficiency, thismechanism may be impaired [52]. The number and affinityof insulin receptors on RBC appear to be related to theglycemic control of participants to study [73]. The number
was lower in poorly controlled diabetics compared to good
controlled diabetics and healthy subjects [73].
ALTERATION IN THE EXPRESSION OF SOME
TRANSPORTERS IN THE RBC MEMBRANE
The transport of many substances through the
RBC membrane, including glucose, is carried out by meansof special structures expressed at the level of cell membrane,transporters. Szabo et al. [63] investigated several RBC
transporters (glucose exchangers GLUT1 (SLC2A1) andGLUT3 (SLC2A3), monocarboxylate transporter MCT1
(SLC16A1), uric acid transporter URAT1 (SLC22A12),cholesterol efflux regulatory protein ABCA1, multidrug
transporter ABCG2, Band-3 protein (SLC4A1) and the active
Ca2+ transporter PMCA4b) and their quantitative expression
in three groups of patients with DM [63]. They reported
significant differences in expression levels of thesetransporters in patients with type 2 DM compared to control
group, but also significant differences in several transporters
among DM patients in the three study groups [63]. A strong
correlation was found between transporter alteration andHbA1c level [63]. The expression level of GLUT1, GLUT3
and MCT1 was significantly lower in the entire group of
diabetic patients compared to the control group, while theexpression of URAT1 was significantly increased [63]. The
expression of the lipid transporter ABCA1 was increased,
while that of the calcium pump (PMCA4b) was decreased indiabetic patients compared to the control group, although
without a statistical significance [63]. For ABCG2 transporterand Band-3 protein no measurable differences werereported between diabetics and control subjects [63]. In thegroup of diabetic patients with complications, a significant
decrease in the level of GLUT1 compared to the control
group and successfully treated patients was found, instead
the levels of URAT1 and ABCA1 were increased in thisgroup relative to the control group and untreated patients[63]. When the transporter expression level was correlatedwith the HbA1c level, it was found that higher HbA1c
concentrations correlated with significantly reduced GLUT1,
GLUT3 and MCT1 expression in the RBC membrane, andincreased URAT1 and ABCA1 levels [63].
Reduced baseline RBC-GLUT1 concentration has
been reported in adolescents with type 1 DM compared tohealthy children [74]. Baseline RBC-GLUT1 was negatively
correlated with HbA1c in diabetic adolescents but not incontrol group [74]. Lowering blood glucose concentration did
not change baseline RBC-GLUT1 in diabetic and control
groups [74], which could demonstrate an impairment of RBC
glucose transport and vulnerability of the brain when facedwith hypoglycemia, considering that GLUT1 is also
expressed in the blood brain-barrier [74]. Hu et al. [75] foundthat the rate of glucose influx through the RBC membrane
was significantly reduced in a group of patients with type 2
DM compared to control subjects, but no significant
difference was found for glucose efflux between the twogroups [75]. One of the explanations for the decreased rateof glucose entry in diabetic RBCs is the change in GLUT1
structure or conformation [75]. According to Hu et al. [75],the altered site could be in the outer domain of thetransporter [75].
CONCLUSIONSHyperglycemia
anddyslipidemia
cause
biochemical changes in the protein structure and lipid
content of the RBC membrane that alter cell membrane
fluidity and RBC deformability. Biochemical changes can be
23

Fiziologia - Physiology • 2023.1(104)followed by changes in cell membrane surface area, cellvolume and shape, and osmotic and mechanical resistance.
Reducing the electrical charge of the membrane surface
makes RBCs more easily lysed by macrophages, and at thesame time less rejected by other RBCs, which may increase
their aggregability and adhesion to the vascular endothelium.
Glycation of hemoglobin, in addition to induced changes in
its secondary structure, can be followed by functional
consequences regarding oxygen affinity and oxygen releaseat the tissue level. Alteration of the secondary structure ofhemoglobin causes a decrease in solubility, increase incytoplasmic viscosity and decrease in flexibility of the RBC
which can ultimately reduce its deformability. The affinity ofhemoglobin for oxygen is altered not only by hemoglobinglycation, but also by changing the concentration of 2,3 DPG,
which can further aggravate hypoxia. Abnormal activity andstructure of Na+-K+-ATPase and Ca2+-APTase, as well asBand 3 protein, may contribute to changes in RBC shapeand volume in patients with DM. Multiple metabolic reactionsin RBC are affected by hyperglycemia. The increase inglucose metabolism via the polyol pathway is followed by theaccumulation of sorbitol and fructose, which in turn, throughthe osmotic effect created, can change the cell volume.
Activation of the polyol pathway alters cellular redox balance,
decreases glutathione, and reduces the antioxidant capacityof RBCs, making cells more vulnerable to the effects ofoxidative stress. In many of the studies, the severity of these
RBC abnormalities was correlated with the level of glycemic
control and the presence of DM complications.
This review of RBC abnormalities associated withDM showed that in this disease several components of RBC
are altered, certainly more than were presented in this paper.
Although the consequences of metabolic imbalances in DM
on RBCs are not completely deciphered, understanding
them helps us to know the effects of hyperglycemia anddyslipidemia on other cells of the body which could pave theway for finding a more effective approach to this condition.
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26

Fiziologia - Physiology • 2023.1(104)MODIFICARI ALE UNOR COMPONENTE ALE ERITROCITELOR IN DIABETUL ZAHARATREZUMAT
Diabetul zaharat (DZ) este o boală metabolică asociată cu multe complicații. Mediul plasmatic hiperglicemic și dislipemic din DZ
determină modificări calitative și cantitative ale membranei eritrocitare, enzimelor, transportorilor și hemoglobinei. Hiperglicemia poate
influența rezistența osmotică a eritrocitelor, activitatea transportorilor și a pompelor din membrana celulară, conținutul de 2,3difosfoglicerat (2,3-DPG), structura secundară a hemoglobinei și afinitatea acesteia pentru oxigen, echilibrul redox intracelular și, incele din urmă furnizarea de oxigen celulelor organismului. Anomaliile biochimice, structurale și funcționale ale eritrocitelor observate în
DZ par a fi legate de starea glicemică a pacienților. Cel puțin la nivelul eritrocitelor, mai multe „ținte” ar putea fi afectate de creșterea
glicemiei, chiar daca această lucrare a trecut în revistă doar câteva dintre ele. Cunoașterea consecințelor hiperglicemiei și dislipidemiei
asupra morfologiei și caracteristicilor funcționale ale eritrocitelor poate fi utilă în înțelegerea efectelor acestor factori asupra altor celuleale corpului și găsirea de terapii eficiente pentru prevenirea acestei boli și a complicațiilor ei.
Cuvinte cheie: diabet zaharat, glicarea proteinelor, metabolismul eritrocitar, transportori, 2,3-DPG

27
Fiziologia - Physiology • 2023.1(104)

POSSIBLE PHYSIOLOGICAL CHANGES INDUCED BY THE PRACTICE
OF PRAYER IN THE CHRISTIAN-ORTHODOX TRADITIONADRIAN SORIN MIHALACHE1,2, ANA-MARIA ZĂGREAN1, DANIELA BĂLAN1, IULIAN STOLERIU3, LEONZĂGREAN1
1 "Carol Davila" University of Medicine and Pharmacy, 050474 Bucharest, Romania
2 Faculty of Orthodox Theology, “Alexandru Ioan Cuza” University of Iași, Romania
3 Faculty of Mathematics, “Alexandru Ioan Cuza” University of Iași, Romania

ABSTRACTIn this study, we follow possible physiological changes induced by the practice of prayer (in the Christian Orthodox Tradition), PCOT,
after a daily individual practice of 30 minutes, for 8 weeks. The participants (n=33) were divided into two groups (Study Group, n=16,
with an average age of 39.2 years and Control Group, n=17, with an average age of 38.6 years). The subjects of the Study Group –faithful Orthodox Christian without the habit of daily prayer being instructed during a 3-hour session on the psycho-emotional, behavioral
and spiritual specifics of PCOT. On subjects in the Control Group did not receive any training. Before and after the 8 week period of daily
PCOT practice, 30 min, saliva samples were collected in the morning, one hour after waking up, pre- and post-test levels of the following
salivary markers: Cortisol, salivary alpha-amylase (sAA), Immunoglobulin A (IgA), Oxytocin, Testosterone and the cytokines interleukin
6 (IL-6) and interleukin 8 (IL-8) being analyzed comparatively. The results indicate a statistically significant decrease in salivary Cortisol
and a post-test increase of IL-6 in the subjects of the Study Group in comparison to the levels measured at pre-test, suggesting that
POCT may improve the physiological response to stress and the inflammatory response. No statistically significant results were obtained
for the other salivary markers. We discuss results in the context of similar studies, highlighting some specific limitations of the studies
that evaluate the physiological changes induced by spiritual/religious experiences, especially those that use salivary markers, while also
formulating some recommendations for increasing the methodological quality of future studies.
Keywords: Christian Orthodox prayer, physiological changes, limitations of studies using salivary markers, Salivary cortisol, Interleukin 6(IL-6), impact of daily prayer, Salivary alpha-amylase (sAA), Immunoglobulin A (IgA), Oxytocin, Testosterone, interleukin 8 (IL-8).

INTRODUCTION
The study of neurophysiological changes
induced by various mental practices with spiritualcontent (such as mindfulness or other forms of
meditation, like compassion meditation), has extended
in recent decades [1], and has significantly diversified [2].
An inventory of Science Citation Index Expanded andSocial Sciences Citation Index using mindfulness or
meditation yielded a total of 19,752 articles publishedbetween 1900 and 2021 [3].
A similar trend is recorded in studies
examining the practice of Christian prayer. A search for
the word "prayer" in PubMed Central, managed by the
National Library of Medicine, provides 77,981 entries, ofwhich 37,912 are studies that mention the term both in
the abstract and in the text as such, since 2019, withmore than 2500 articles published every year1.
Numerous studies on prayer focus on theevaluation of the therapeutic potential, significant resultsin the effort to face stressful demands (coping) [4], in
improving the mood of doctors and patients [5], in pain
management [6], in improving the condition of patients
undergoing surgery [7] or in improving cardiovascular
reactivity [8] being reported. Two meta-analyses, carriedout in the last decade, report correlations between thepractice of prayer and the improvement of mental health[9], but also with the school results of students [10].
The present research relies on the generalhypothesis that the individual practice of prayer in theChristian-Orthodox tradition (PCOT) results inphysiological changes. We aimed to highlight this bymeasuring the changes in some salivary markers. For
this, we chose markers commonly used to assess thephysiological response to stress, in various
psychological and psychopathological conditions(anxiety, depression), but also to evaluate theeffectiveness of some therapeutic interventions,
including Spiritual/Religious experiences (S/RE)(mindfulness, compassion meditation, self-compassion).
PHYSIOLOGICAL PARAMETERS - DEPENDENTVARIABLESIn the present study, the independent variableof the task (experimental task) is the practice span ofPCOT, for 30 min. daily, for 8 weeks.
Salivary cortisol is an important biomarker in identifying
the body's reaction to stress. The cortisol release ismediated by the hypothalamic-pituitary-adrenal (HPA)

Received 22nd of January, 2023. Accepted 26th of February, 2023. Address for correspondence: Adrian Sorin Mihalache "Carol Davila"
University of Medicine and Pharmacy, 050474 Bucharest, Romania. Faculty of Orthodox Theology, “Alexandru Ioan Cuza” University of Iași,Romania. Email: admiso@gmail.com

28

Fiziologia - Physiology • 2023.1(104)axis, through corticotrophin-releasing hormone (CRH)
produced in the hypothalamus [11]. As one of the mainstress hormones, cortisol is an important marker inassessing the impact of S/RE.
Salivary alpha-amylase (sAA) is an indicatorof sympathetic stimulation and the presence of acuteand chronic psychosocial stress [12], being closely
related to stress-induced changes in the SNS [13].
Recent studies have confirmed the influence of mental
states, such as relaxation or stress, on the level of sAA[14].
Immunoglobulin A (IgA) plays a role inmucosal(humoral)immunity,
preventing
microorganisms from entering the body through themucosa [15]. Being an indicator of immune activity, thisbiomarker was included in the list of analyzed dependent
variables, as it is also used to highlight the response tostress [16]. The fact that distress modulates IgA releaseby B cells, decreasing their level [17], provides reasonsfor using IgA in the assessment of possible S/REinduced changes.
Oxytocin modulates social behaviours andcognitions [18], the activation of oxytocin networks,
along with the activation of the vagus nerve, being partof the physiological adaptation of pro-social behaviour[19]. Oxytocin plays a particularly important role indisplaying emotional attachment, in the parent-infant
relationship through contact and interaction [20],regardless of cultural and gender variations [21]. Since
PCOT also includes content with pro-social andempathy-cultivating meanings, in the present study wealso included oxytocin among the dependent variables.
The level of testosterone is reported inexperiences of cognitive-emotional significance, innavigation tasks, in spatial cognition [22], in memory andlearning [23]. High levels of testosterone have also beenreported in elderly subjects, who show increased
cognitive performance [24], while low testosterone
levels may cause low sleep quality [25] and increased
empathy [26]. It is known that testosterone levels are
determined by sexual differences, by some personality
traits and aspects related to emotional control [27]. The
inclusion of testosterone in the package of dependent
variables in the present study is justified by the fact that
correlations between the testosterone level and thelevels of stress [28], depression and anxiety [29],empathy [30] have been reported.
The cytokines interleukin 6 (IL-6) andinterleukin 8 (IL-8) are markers of the degree of
local/systemic inflammation and correlate with thedegree of activation of the autonomic nervous systemand the hypothalamic-pituitary-adrenal axis. Interleukin6 intervenes in the response to somatic stress [31],being present, together with pro-inflammatory cytokines,
in the pathophysiology of depression [32].
Acute psychosocial stress, increasing IL-6
levels for a relatively long period, suggests that
sympathetic activity and cortisol release are involved in
the increase in salivary IL-6 [33]. IL-8 also plays a role in
inflammatory mechanisms [34], anxiety [35], depression[36] and chronic stress [37]. As they are closely related
to the stress response, IL-6 and IL-8 were included
among the dependent variables in this study.
(Since we aim to highlight whether theintervention in the form of an individual practice of PCOT
causes, after 8 weeks of daily practice, for 30 min.,changes in the mentioned physiological parameters, wecall these parameters dependent variables.)
WORKING HYPOTHESESIn the present study we aimed to evaluate if thepractice of PCOT, for 8 weeks, with practice intervals of
30 minutes daily (variables independent of the task),
causes a decrease in the levels of sAA, salivary
testosterone, salivary cortisol, IL-6 and salivary IL-8, as
well as an increase in salivary IgA and Oxytocin(dependent variables).
The working hypotheses were:
Hypothesis 3.1 - PCOT for 30 min./day, for 8 weeks,causes a decrease in the levels of salivary Alpha
Amylase (sAA);
Hypothesis 3.2 - PCOT for 30 min./day, for 8 weeks,causes an increase in salivary Immunoglobulin A (sIgA);
Hypothesis 3.3 - PCOT for 30 min./day, for 8 weeks,causes a decrease in salivary Cortisol (SC) levels;
Hypothesis 3.4 - PCOT for 30 min./day, for 8 weeks,causes a decrease in salivary Testosterone levels;
Hypothesis 3.5 - PCOT for 30 min./day, for 8 weeks,causes an increase in salivary Oxytocin (SO) levels;
Hypothesis 3.6 - PCOT for 30 min./day, for 8 weeks,causes changes in levels of Interleukin 6 (IL-6 sal);Hypothesis 3.7 - PCOT for 30 min./day, for 8 weeks,causes changes in salivary interleukin 8 (IL-8 sal) levels
Following these working hypotheses, wechecked if there were statistically significant differences
between the central values measured for the Study
Group (SG) and the Control Group (CG), pre- and posttesting, for each of the seven variables: sAA, sIgAs, SC,
Salivary Testosterone, Salivary Oxytocin (sO),
Interleukin 6 (IL-6 sal) and Interleukin 8 (IL-8 sal). To test
the hypotheses, we compared the values obtained fromthe analysis of saliva samples collected from subjects in
SG and CG, intra-group and inter-group, pre- and posttest.

29
Fiziologia - Physiology • 2023.1(104)PARTICIPANTS
The study included a group of 33 healthy adults
recruited through online recruitment notices.
The 33 subjects (24 women, 9 men, aged 20-57
years, the average age being 38.9 years) were divided
into two groups (CG Control Group = 17 participants, 12
women and 5 men, the average age of 38.6 years; Study
Group SG = 16 participants, 11 women, 5 men, theaverage age of 39.2 years). The division into the twogroups was made on the basis of a directed interview,
with the following criteria: people who live in Bucharest,with no experience in PCOT, no history of brain
disorders, non-smokers, no weekly volunteer practice(more than 8 weeks, in the last half a year), without
chronic conditions and who do not follow drug
treatments.

To make up the study group, for the participantsinterview, three additional criteria were introduced,
regarding the independent variable:- subjects should know the general
information on the Christian-Orthodox religion andspirituality (declared membership to Orthodox
Christianity);- subjects should not be constant (daily), but
only occasional PCOT practitioners;- subjects should be willing to fulfill therequirements of the experimental task (individual
practice of PCOT, daily, for 30 minutes, over a period of
8 weeks). The composition of the two CG and SG
groups followed a similar gender and age structure tohave a better control of the effects of random variables.

Table I.
Age and gender structure of CG and SG, specifying contaminated samples (c). The numbers in the table represent subject
coding.
cod
25
23
6
12
13
2
27
5
26
3
29
4
16
14
15
9
30

30

Study Group (SG)sal gender
✓
F
✓
F
✓
F
✓
F
✓
F
✓ (c)
F
✓
F
✓
F
✓
F
✓
F
✓
M
✓ (c)
M
✓
M
✓
F
✓
F
✓
M
✓
M

age
42
21
41
57
33
37
39
51
35
40
37
41
32
48
41
26
47

o age
45
20
38
56
30
38
39
49
34
42
38
42
32
46
42
27
47

Control Group (CG)gender
sal
✓
F
✓
F
✓
F
✓
F
✓
F
✓
F
✓
F
✓
F
✓
F
✓
F
M
✓ (c)
✓
M
✓
M
✓
F
✓
F
✓
M

cod
28
7
10
22
19
20
17
8
24
18
31
21
32
11
33
1

Fiziologia - Physiology • 2023.1(104)RESEARCH STAGES, DIAGRAM AND TIMELINEStage I (weeks 1-2) – Signing the consent
statement. After the preparatory stages, which included
the approval of the Ethics Commission of UMPCD andthe formation of the SG and CG, this stage included
providing information regarding the general frameworkof the study and the signing of the declaration of
informed consent by the subjects of the SG and CG.
Stage II (week 3) - Pre-test measurements (preexperiment), provided for the collection of saliva
samples from SG and CG subjects, randomized, tomeasure the concentrations for the mentioned markers.
Stage III (week 4) – Presentation of psychoemotional and cognitive-behavioural coordinates of
PCOT for SG subjects. SG participants watched apresentation (3.5 hours) on the independent variable (2hours of presentation based on a PPT and 1.5 hour of
dialogue with an expert). (The support was compiled andpresented by Adrian Sorin Mihalache, graduate of
Orthodox theology, lecturer at the Faculty of Orthodox
Theology in Iași, relying on some texts by well-known
authors, included in The Philokalia). The presentationconsisted in highlighting the emotional, cognitive andspiritual psycho-behavioural aspects of PCOT andpractical indications, namely: the role of attention in
prayer, the importance of the inner mood for theexperience of prayer, individual preparation measures,
recommended before the experience of prayer,supervision of daily behavior, for a suitable mood for
prayer, establishing the program/contents of personal
prayer, for the 30 min. of daily practice, in the case ofeach SG participant, through the spiritual counselingprovided by a Christian Orthodox priest.
Stage IV (weeks 5-12). The experimental
intervention for subjects in SG. At the start of testing(experimental interval), SG subjects were given, in
addition to the Phase III exposure, PCOT
recommendations to follow: choose a quiet place, with
low noise, the 30-minute interval should not be
fragmented, avoid hasty reading of the prayer texts,
carry out the activity carefully. The experimental intervalfollowed. For 8 weeks, the subjects in the SG practiced
30 minutes PCOT, individually, at home, (5.5hours/week), keeping in touch by email and phone witha person from the research team and with the priest.
Stage V (weeks 13-15). Post-test
measurements, in which saliva samples were collectedagain, as in stage II, one hour after waking up in themorning.
Stage VI followed, intended for the processing
and interpretation of primary data.

SAMPLING CONDITIONS AND PREPARATION OF
SALIVA SAMPLES
The collection was done in the morning, onehour after waking up, one week before the experimental
interval (PCOT practice for SG subjects) and finally after
the experimental intervention, a single sample, for eachof the SG and CG subjects, in the morning, one hour
after waking up. Post-testing, saliva sample collection
from SG and CG subjects took place in threeconsecutive days, and SG subjects continued individual
PCOT practice until the day of collection.
The advantages of determining salivary
parameters also consist in the fact that collecting saliva
does not involve discomfort (compared to taking blood
samples) and, being non-invasive, does not involve
procedural difficulties, and can be easily applied instudies focusing on groups of subjects. In addition, since
the collected samples can be stored in arefrigerator/freezer, without the risk of deterioration orsignificant changes in concentrations, it is possible toproperly analyze and obtain the values of theinvestigated salivary parameters.
For the collection, the volunteers traveled fromhome to the laboratory headquarters. The subjects werealso trained on how to collect saliva, and used sterile
Salivette sampling vials (Sarstedt, Italy).
The collection instructions were: no alcohol andcoffee beforehand, for at least 12 hours; no foodconsumption for at least 8 hours prior; no liquid
consumption 1 hour before collecting; no physicalexertion on the morning of the collection; no smoking forat least 12 hours before collecting; avoid tooth brushing
the night before and on the morning of collection, but atleast 2 hours before saliva collection (as other oral
hygiene maneuvers are not allowed - dental floss,
mouthwash, simple brushing); collection of unstimulated
mixed saliva in sterile vials, kept on ice; collection donefor 10 minutes (the saliva from the first minute could bediscarded); if the sample volume was less than 3 ml, thecollection was repeated after a 10-minute break. The
subjects were in a relaxed, sitting position, did not
swallow during collection; no communication with otherpeople was allowed.
Immediately after collection, the saliva samples
were placed in ice boxes and transported in an
isothermal envelope, then centrifuged cold for 20
minutes at 6000 rotations/minute. From the time ofcollecting to centrifugation, the time interval was a
maximum of 3 hours.
Each supernatant sample was stored in sterile 1.5
ml Eppendorf tubes and stored at –20°C for up to 30
days.

31
Fiziologia - Physiology • 2023.1(104)BIOCHEMICAL DETERMINATIONS
The monitored parameters were dosed in the
supernatant samples, according to the working protocol
provided by the manufacturer of each specific reagent
kit.
All the obtained results were related to salivary
albumin concentration, to remove the effect of salivary
flow variations. Salivary albumin was determined by the
ELISA technique, using a reagent kit from Diametra(Italy) and a STAT FAX 303 semi-automatic ELISA
analyzer (USA). Thus, parameter values wereexpressed in units/mg albumin.
(a) α-Amylase - The activity of this salivary
enzyme was determined on an automatedbiochemistry analyzer, A25 – Biosystems(Spain), in absorbance/colorimetric mode,using a specific reagent kit for α-amylase (αAmylase Direct, 5 x 20 ml) from the same
company. Results were expressed in U/mg
albumin.
(b) Immunoglobulin A (Ig A) - Salivary IgA
concentration was determined by the ELISA
technique, using a kit of reagents from
Diametra (Italy) and a semi-automatic ELISA
analyzer STAT FAX 303, USA. Results wereexpressed in mg/mg albumin.
(c) Cortisol - Salivary cortisol concentration wasdetermined by the ELISA technique, using a
reagent kit from Diametra (Italy), and a semiautomatic ELISA analyzer STAT FAX 303,
USA. Results were expressed in ng/mg
albumin.
(d) Testosterone - Salivary testosterone
concentration was determined by the ELISA
technique, using a kit of reagents from
Diametra (Italy) and a semi-automatic ELISA
analyzer STAT FAX 303, USA. Results wereexpressed in nmoli/g albumin.
(e) Oxytocin - The salivary concentration ofoxytocin was determined by the ELISA
technique, using a reagent kit from Detect X(USA), and a semi-automatic ELISA

32

(f)

analyzer STAT FAX 303 (USA). The results
were expressed in pg/mg albumin.
IL-6 and IL-8 - Salivary concentrations of IL6 and IL-8 were determined bychemiluminescence, on an automatic
analyzer IMMULITE 1000, Siemens(Germany), using specific reagent kits from
Siemens (Germany). The results wereexpressed in pg/mg albumin.

TESTING THE WORKING HYPOTHESES
Following the specific measurements for eachparameter, we analyzed the data from a statistical pointof view, after excluding the data provided bycontaminated/improper samples.
We shall next present the values measured in
the saliva samples taken from the CG and SG subjects,
which correspond to salivary Cortisol and IL-6, for which
the obtained results are statistically significant.
We aimed to highlight the possible changes thatappear among the subjects in the SG, by measuring thevariables mentioned before and after the test. To test
Hypotheses 1-6, in a first phase we performed a pair t
test. Also, working hypothesis testing was performed
with the Wilcoxon sing-rank Test, by comparing themedians of two populations using two adequate samples.
The test assumes that the two samples are paired, sothey are not independent. To test Hypotheses 1-6, in a
first phase we performed a t test for paired data. This
test checks the equality of the mean values of thementioned variables, pre and post-test. However, since
the number of samples is relatively small and theobserved values are not necessarily normally distributed,
the result of a t test in this situation can only be indicative.
Therefore, the working hypotheses were also tested with
the Wilcoxon signed-rank test. This test is the nonparametric variant of the paired data t-test, used tocompare the medians of two populations using two
paired data samples. In contrast to the t-test for paired
data, the Wilcoxon signed-rank test does not require thenormality of the data to be applied. We also mention thatboth the mean and the median are measures of thecentral tendency of the data.

Fiziologia - Physiology • 2023.1(104)To test the working hypotheses, regarding the evolution of SG in the experimental interval, the Wilcoxon test was applied - for all seven chosen variables, we have:
The null hypothesis = the median value of the pre- and post-test differences is 0,
Alternative hypothesis 1 = the median value of the pre- and post-test differences is less than 0 (for a left-sided test),
Alternative hypothesis 2 = the median value of the pre- and post-test differences is greater than 0 (for a right-sided test), or
Alternative hypothesis 3 = the median value of the pre- and post-test differences is non-zero (for a two-sided test).
Each test returns the P value, as shown in Table II. At a significance level of 0.05 (5%), a P value of less than 0.05 will cause the null hypothesis to be rejected in favor of theappropriate alternative.

Table II. The values obtained with the Wilcoxon Test for each of the medians of the 7 variables(Salivary α-Amylase, Salivary IgA, Salivary Cortisol, Salivary Testosterone, Salivary Oxytocin, Salivary Interleukin 6 and Salivary Interleukin 8
obtained for Study Group (SG) subjects, pre- and post-testing

α-Amylase

Ig A

Cortisol

Testosterone

Oxytocin

IL-6

IL-8

Wilcoxon test (left)

0.26220.6816

0.98980.56040.07420.0183*0.0778Wilcoxon test (right)

0.75560.32930.0113**0.45070.93030.983

0.9264Wilcoxon test (both)0.52450.65860.0226***0.90150.14840.0366***

0.155633
Fiziologia - Physiology • 2023.1(104)RESULTS
As shown in Table II, in the case of two of the studied variables, we obtained statistically significant results. It is about
salivary Cortisol (s C) and Interleukin 6 (IL-6)2.
SALIVARY CORTISOL
The median values of salivary Cortisol concentrations measured in SG subjects in post-testing were statisticallysignificantly lower (15.77) compared to the median values in pre-testing (18.10). The result validates hypothesis 3.3 which
aimed to decrease the level of sC as a result of PCOT practice among SG, for 8 weeks, 30 min/day.
Subject
code

2
3
4
5
6
9
12
13
14
15
16
23
25
26
27
29
30
med valdev stt testgender

F
F
M
F
F
M
F
F
F
F
M
F
F
F
F
M
M

S1 CORTISOL
ng/mg
albumin

24,33
17,56
22,9
22,34
19,33
16,8
14,87
15,11
17,65
15,78
17,89
12,87
17,68
18,9
17,56
18,10
3,12
0,022

S2 - CORTISOL
ng/mg albumin

27,12
16,42
14,04
17,89
12,57
17,54
12,31
15,87
13,29
15,54
18,26
12,21
17,31
9,9
16,42
15,77
4,00

Table III. Measured values for Cortisol, Pre-test (S1) and post-test (S2), after the experimental intervention (8 weeks of PCOT
practice), in the subjects of the Study Group (SG).

Fig. 1. Graphic representation of the measured values for Cortisol, pre- (S1) and post-test (S2), after the experimental intervention (8weeks PCOT practice), for the 15 subjects in the Study Group (SG)34

Fiziologia - Physiology • 2023.1(104)INTERLEUKIN 6 (IL-6)
For SG subjects, the median pre-test IL-6 level (14.37) is lower than the post-test median (15.28). The difference isstatistically significant (p=0.05), validating hypothesis 3.6 - the increase in IL-6 levels, induced by PCOT practiced for 8 weeks.
Subject
code

Gender

2
3
4
5
6
9
12
13
14
15
16
23
25
26
27
29
30

F
F
M
F
F
M
F
F
F
F
M
F
F
F
F
M
M

med valdev stt testS1 - IL-6
pg/mg
albumin
15,4
15,8
12,7
15,8
14,5
12,9
13,5
15,4
12,7
11,9
12,3
16,6
16,7
14,5
14,9

14,37
1,59
0,034

S2 - IL-6
pg/mg
albumin
16,3
12,5
14,9
16,9
16,4
15,9
12,2
17,5
16,9
12,7
12,9
17,1
16,4
15,8
14,9

15,28
1,84

Table IV. Values measured for IL-6, Pre (S1) and post-test (S2), after the experimental intervention (8 weeks of PCOT practice), for the
15 subjects in SG.

Fig. 2. Graphic representation of the measured values for IL-6, pre- (S1) and post-test (S2), after the experimental intervention (8weeks PCOT practice), in SG subjects.

35
Fiziologia - Physiology • 2023.1(104)Wilcoxon rank sum test for Study Group medians and Control Group medians, post-test (P-values)
α-Amylase

Ig A

Cortisol

Testosterone

Oxytocin

IL-6

IL-8

Wilcoxon test (left)

0.61420.71240.64560.33150.22100.94500.8302

Wilcoxon test (right)

0.40170.30190.37000.68350.79110.05980.1805Wilcoxon test (both)

0.80340.60380.74000.66290.44200.11950.3611

Table V. Wilcoxon test for comparing the median values of SG and CG
For the testing of the secondary hypotheses (3.1.1, 3.2.1, 3.3.1, 3.4.1, 3.5.1, 3.6.1, 3.7.1), aimed at the comparative analysis of SG and CG pre- and pro-testing, we apply the Wilcoxon
test. For each of the mentioned variables, we have:
Null hypothesis = the median value of the pre- and post-test differences is 0,
Alternative hypothesis 1 = the median value of the pre- and post-test differences is less than 0 (for a left-sided test),
Alternative hypothesis 2 = the median value of the pre- and post-test differences is greater than 0 (for a right-sided test), or
Alternative hypothesis 3 = the median value of the pre- and post-test differences is non-zero (for a two-sided test).
(Each test returns the P-value, shown in Table V. At the 0.05 (5%) significance level, a P-value less than 0.05 will cause the null hypothesis to be rejected in favour of the appropriate alternative.)
The Wilcoxon test reveals that there are no statistically significant differences between the central values for CG and SG, pre- and post-test, for any of the mentioned variables, whichmeans that none of the subsequent hypotheses formulated (3.1.1, 3.2.1, 3.3.1, 3.4.1, 3.5.1, 3.6.1, 3.7.1).
Regarding the other variables, sAA, IgA, sO, sT, IL-8, the formulated working hypotheses were invalidated.
COMPLEMENTARY HYPOTHESIS TESTING
In the second phase, for the subsequent hypotheses, we analyzed the differences between the values measured in the SG subjects and those recorded in the CG subjects, pre and posttesting. This allowed the comparative analysis of the evolution of CG and SG in the experimental interval of 8 weeks, pre and post-testing. We tested whether there were statistically significant
differences between SG medians and CG medians, for each of the seven measured variables.
We used the Wilcoxon test, which tests the null hypothesis (that is, the two samples from continuous distributions have equal medians, compared to the alternative of not being equal),
taking into account that the two samples (for SG and for CG) are not independent.
For this purpose, we used Wilcoxon's rank sum test, which tests the null hypothesis: (H0): the two samples are drawn from continuous distributions having equal medians, with thealternative (H1): the medians of the two populations are not equal. We must mention that, instead of the t test for the equality of the means of two independent populations, we again used a nonparametric test, this time the Wilcoxon rank-sum test or Mann Whitney U test, because the researched samples for SG and for CG do not have large volumes, which means that the t test cannot
be applied in this case either.
The obtained results (Table V - Wilcoxon test for comparing the median values of SG and CG) show that there are no statistically significant differences between the central values of SG
and CG, pre and post-testing36

Fiziologia - Physiology • 2023.1(104)DISCUSSIONSIn the last decades, studies have followed the changes
produced by spiritual/religious experiences (S/RE) at thephysiological level, measuring the level of some biologicalvariables, which are also health indicators, such as cortisol(blood or saliva) or cytokines IL-6 and IL-8.
In what follows, we shall discuss the results of thepresent study in the context of results reported by similar
studies that did not have experimental PCOT tasks.
SALIVARY CORTISOL
In the present study we identified a decrease insalivary Cortisol levels among SG subjects, post-test, after
the 8 weeks of PCOT practice. The median of the pre-testvalues was 18.10, and the median of the post-test values
was 15.77.
Other studies have also reported a reduction insalivary cortisol as well. One of them focused on thepractice of mindfulness (in the case of a study groupconsisting of war veterans) [38], and another with thepractice of yoga-nidra exercises as an independent
variable of the task, in patients suffering from insomnia[39]. Another study suggests that the adoption ofreligious behaviour may be a predictor of the pattern of
diurnal salivary cortisol levels over a 10-year period. The
study was conducted on a sample of 1,470 adults from the
USA who provided reports on religious participation, withresults showing that more sustained participation inreligious experience correlates with lower diurnal salivary
cortisol levels [40].
However, there are also studies that question the
reduction of salivary Cortisol levels by S/RE. The study
that reports inconclusive results in the case of yoga
exercises practiced for 10 weeks (one hour/week) by agroup of veterans affected by Post-Traumatic Syndrome[41] is revealing. Another study followed the impact ofpracticing yoga breathing exercises (Pranayama) - 3 times
a week, with durations of 45 min., for 6 months - by a groupof young adolescents, pointing out that after theexperimental interval the subjects did not register a lower
salivary cortisol level compared to the cold pressor test
[42].
The fact is significant, in the context of the otherresults. However, we should underline that in most of thestudies discussed here, the sampling was done according
to more complex schemes, which provided for threesamplings (on waking, 30 minutes after waking up and at
bedtime) [43] and even four samplings in the same day (inthe case of sC level testing, in the cold pressor test

situation, during the cold pressor test, respectively 20, 40
and 60 minutes after the experience) [44], 4 times a day(immediately after waking up, 30 min. after waking up,
before lunch and bedtime) [45], but also 5 times a day(immediately after waking up, 30 min. apart from waking
up, at lunch, dinner and bedtime), in two consecutive days
[46].
Therefore, an important drawback of the present
study was that the saliva collection did not take into
account the recommendations regarding the standard
collection protocol - which provides better data on salivary
Cortisol levels, and which recommends several collections
throughout the day, for several days [47]. For salivary
Cortisol, for example, an experimental design that allows
for event-centered collection and measurement sessions(Event-Related Design) with others centered on frequency
and duration (Time Based-Design) would be required [48].
At the same time, we should also take into account thatsuch an experimental design, which also includes thedemands of daily practice, over long periods, could bedemanding for the volunteer participants.
On the other hand, it can be seen that thedecrease in the level of salivary Cortisol could becorrelated with the decrease in anxiety assessed by the
Hamilton Scale.
INTERLEUKIN 6 (IL-6) IN SALIVA
This marker could be relevant for the present
study as well, since in our case the SG subjects registeredan increase in the level of IL-6, from 14.37 to 15.28.
Two other studies highlight increased IL-6 levels.
One of them reported that yoga practice caused anincrease in IL-6 levels, in the case of a group of 38 people(19 men, 19 women) included in a program of spiritual
practices in a retreat, consisting in yoga exercises andmediation (totaling approx. 5 hours/day and a vegetariandiet) [49]. Another study reported an increase in IL-6 withan MBSR (Mindfulness Based Stress Reduction)intervention in a group of lonely elderly subjects (65-85),
with a lower level of immune activity. An increase in IL-6
levels was noticed after 8 weeks of MBSR sessions,indicating that mindfulness training may help counteract
the effects of aging, as immunocompetence declines withage.
However, in the present study, the subjects have amuch lower average age, so that other studies would benecessary, with a selection of subjects according to stricter
age criteria, and with the prior evaluation of the level of
immunocompetence, to highlight possible similar effects.
37

Fiziologia - Physiology • 2023.1(104)At the same time, we must highlight the fact that
there are studies reporting a decrease in the level ofsalivary IL-6 after S/RE invoice interventions, bycultivating self-compassion [50], by mindfulness (IBM) [51],including the case of people with alcohol addiction [52],but also through the practice of yoga, for 8 weeks [53].
Other studies note the lack of effectiveness of some ES/R
practices for lowering the salivary IL-6 level, such as thecase of MBCT (Mindfulness Based Cognitive Therapy), in
the case of cervical cancer patients [54] or in the case of
MT sessions (Transcendental Meditation) [55].
These results indicate the need for further studies
to confirm the results on how PCOT changes the level ofsalivary IL-6.
INTERLEUKIN 8 (IL-8) IN SALIVA
In the present study, we did not underline a change
in the IL-8 level among SG subjects by PCOT, the working
hypothesis 3.7 being invalidated.
Other studies obtained changes in the IL-8 level
by S/RE practices. One of these highlighted the increaseof IL-6, in the case of the group of 38 subjects practicing
yoga (retreat, yoga meditation 5 hours/day and avegetarian diet). In the same study, an increase in IL-8,
along with IL-10, and a decrease in IL-12 are also reported[56]. Another study, (n = 95, healthy people, aged 18 to 67
years), showed a decrease in IL-8 after 3 days of
meditation compared to 3 days of vacation, along with adecrease in IL-8 -6 and an increase in IL-12 [57].
One of the reasons why the present study did notreveal changes in IL-8 by PCOT practice could be related
to the conditions of saliva sample collection. In the studies
mentioned in the discussions here (focused on the yoga
retreat and 3 days of meditation), the sampling was done
according to a more complex protocol than in the present
study: immediately after waking up (4:45 AM), 30 min.
after waking up (5:15 AM), at 9:15 AM at 12:30 PM, before
lunch, at 5:10 PM during a break and, finally, at 8:30 PM
after meditation, just before going to sleep [58] .
In the subsequent part, we shall also look into
other studies, with obtained significant results regarding
the changes induced by S/RE practices at the level of thesalivary markers on focus, but which had more complex
and precise collection protocols, an aspect that could be
an important cause for the results of the present study.
SALIVARY ALPHA-AMYLASE - SAA
In the present study, we failed to outline anychanges in sAA in SG subjects, through PCOT, for 8
weeks, working hypothesis 3.1 being invalidated.
38

Other studies have reported decreases in sAA
among subjects that practise meditation. In a study
focusing on mindfulness - MBI (Mindfulness Based
Intervention), participants (n=28), after 3 months ofpractice (4 sessions of three hours, every two weeks),
reported decreases in sAA and salivary Cortisol, after theintervention [59]. Another study, taking into account 2
working groups (study and control) consisting of 9 healthy
students (interns in veterinary surgery, one group being
the study group), reported changes in salivary Cortisol
levels after the intervention (5 minutes of focused
breathing, practised before a surgical intervention
performed by intern physicians), but did not identify
statistically significant changes in salivary α-Amylase [60].
Another randomized study (n=57), with subjects
experiencing sleep disturbances after successful cancer
treatments, highlighted the effectiveness of an intervention
based on strengthening the mind-body connection,measuring, among other things, the level of sAA. One of
the 3 groups of participants benefited from a sleep hygiene
program (n=18), the other two groups participated in a
meditation program focused on the mind-body connection(Mind-Body Bridging - MBB, n=19) and, respectively,
Mindfulness Meditation (n=20), with 3 sessions (1/week),for 2 consecutive weeks. Pre-test and post-test
measurements (one week after the last session) revealed
lower average levels of sAA (wake-up sample) for MBB
meditation practitioners, compared to those who
participated in the program of sleep hygiene [61].
Another study looked into possible correlations
between the quality of meditation (degree of concentration,
length of time since practicing meditation) and the level ofsAA in waking state, in the case of 77 expert subjects(average age = 58 years). Four saliva samples werecollected in successive moments - immediately after
waking up, at 15 min., 30 min. and respectively 45 min.
from the moment of waking, before engaging in sports
activities or eating. Measurements also suggested a lower
level of sAA immediately after waking in the case of people
with more meditation experience who reported deeper
meditation sessions [62]. Finally, another study highlighted
that mindfulness-based intervention (MBI – Mindfulness
Based Intervention) can modify perceived stress and thelevel of activation indicators of the SNS (salivary alphaamylase, sAA) and the HPA axis (salivary cortisol, sC).
The participants were separated into two groups,
according to the voluntary option, 28 of them participating
in an MBI program for a period of 3 months, the other 46
forming CG. Comparative analysis of primary data(measurements of attention level, self-perceived stress,
Fiziologia - Physiology • 2023.1(104)level of sAA and sC), that were collected pre- and posttesting, revealed low levels of sAA in SG members,compared to the latter CG subjects, the latter registering
post-testing increases in values [63].
The fact that in the present study no change in sAA
was revealed may be due to the adopted measurement
protocol. In the studies mentioned here for discussion,
several saliva samples were collected at shorter time
periods immediately before and after the experimental
task (10 min. and 0 min. before a surgical intervention and
10 min. after the surgical intervention) [64]. In anotherstudy, saliva samples were collected at several timeintervals after waking up: 30, 150, 480 and 720 minutes
after waking up, with the last measurement taking place
before going to sleep [65]. On the other hand, in anotherstudy, the investigated subjects were experienced
meditators, and the sampling was made immediately after
waking. These aspects are important, and in the present
study the sampling was done by collecting a uniquesample for each subject, at the end of an 8-week interval,
and at a distance of almost an hour from the moment of
waking up (with subjects taking approx. 30 min. to get tothe laboratory), and after an interval of maximum 18 hours
from the experimental task, circumstances that could haveinfluenced the quality of the data.
IGA ANTIBODIES IN SALIVA
In the present study, we did not identify changes
in SIgA through PCOT practice for 8 weeks, 30 min. daily,
by SG subjects, working hypothesis 3.2. being invalidated.
Other studies have shown an increase in the levelof SIgA by S/RE. Thus, a study focused on a number of 24
participants, reporting increases in SIgA after 6 weeks of
MBSCI - Mindfulness Based Self Compassion Intervention
exercises (2 hours per week, with a total of 12 hours ofpractice) carried out at work [66]. Two other studies show
increases in IgA after 16 weeks of yoga practice [67], andrespectively after only 90 minutes of practice, in the caseof a group of 23 adult women who practiced yoga
meditation [68]. Another study found an increase of SIgA
in the case of 31 subjects (28 women and 3 men) after 2hours of weekly mindfulness and self-compassionintervention sessions over 2 months [69]. Finally, anotherstudy showed the effectiveness of online MBSR sessions(30 min./ week, for 2 months), in the case of 22 subjects,
with saliva measurements showing an increase in SIgA
post-meditation [70].
The absence of results in the case of the present
study could also be caused by the protocol for collecting.

In the mentioned studies, saliva samples were collected
immediately after the experimental intervention (yogapractice [71], meditation [72]), or – for comparative
analyses, before and after the experimental intervention[73], before and after the sessions that took place in thecollecting place [74]).
The experimental design in the present study took
into account usual PCOT practice, which recommends
praying in a private space, not in the laboratory, which
resulted in the sampling being done one hour after waking
up at the remote project location, with a span of 10-20
hours possibly having passed since they completed thedaily task, the 30 min interval. (as the experimental design
did not fix an identical area for all subjects for PCOT).
SALIVARY TESTOSTERONEThe present study did not reveal changes in the sTlevel among SG subjects after PCOT practice for 8 weeks,
the 3.4 hypothesis being invalidated.
Several studies have reported changes in the sTlevel, as a result of S/RE practices, but also in the case ofcontrol subjects in such profile studies. One study focusedon a group of 10 men (age 26.3 ± 2.5 years) with noexperience in yoga practice, provided 90-minute practicesessions, saliva samples being collected before andimmediately after this experimental interval, but also at 60
minutes and 120 minutes after the yoga exercise [75],showing an increase in testosterone levels after 120
minutes after the yoga session. Another study reported anincrease in testosterone levels among a group of students
who practiced non-physically active relaxation techniques(Body-scan meditation and Mindfulness Meditation),compared to other relaxation techniques that involved
physical activity [76 ]. Finally, another study looked atstudents who practiced MBSR (2 hours/week, for 11
weeks), with volunteers providing two saliva samples (inthe morning and evening), before the start of the course
and at the end of the activity. The study reported a different
result from those mentioned so far, namely a significant
increase in testosterone levels in the morning samples, in
CG, in women compared to meditative participants andbasic hormone levels, suggesting that testosterone may
increase as a result of stressful situations [77].
As the PCOT experience engages specificspiritual relationships, cognitive activities, and emotional
patterns, it is possible that prayer, practiced over a longer
period of time, influences testosterone levels, showing
relevance in assessing S/RE impact. However, as wehave seen, the present study failed to show statistically39

Fiziologia - Physiology • 2023.1(104)significant changes in testosterone levels. In this case as
well, the collecting protocol could be one of the causes forthe inconclusive results, as the collecting was carried out
only once a day, at a considerably longer time interval than
the individual experience.
SALIVARY OXYTOCIN
Also regarding the salivary oxytocin (sO), thepresent study did not obtain statistically significant
changes among SG subjects in post-testing compared to
pre-testing.
Other research, however, has highlighted suchchanges. A recent study reported that mindfulness-based
interventions (MBIs) could be effective in increasing
empathy among healthcare professionals, based on sO.
Taking into account a total of 68 volunteers, psychology
students divided into two groups, SG and CG, the study
revealing an increase in the level of sO. SG subjects (n =42) benefitted from a mindfulness session, while CG
subjects (n = 26) did an emotion recognition exercise andother creative activities. Comparison of the data showedthat the MBI session corresponded to a significant
reduction in negative affect (d = -0.56, p < 0.001) andstatus anxiety (d = -0.54, p = 0.007) in SG, along with anincrease in sO (d =0.99, p < 0.001), relative tomeasurements obtained from CG subjects [78]. In the
study, saliva samples were collected 10 minutes before
and 10 minutes after the experimental intervention. We
also find in this study, as in the case of the previous ones,
elements that suggest revisions in the experimental
protocol for future studies aimed at evaluating PCOT.
At the same time, there are studies that place
S/RE and the level of sO in a different relationship,
identifying a possible correlation between increased levelsof sO and the manifestation of distrust in
spirituality/religiosity, as well as critical beliefs about
religion, compared to those with low levels by Bone [79].
Results of this kind support the need for future evaluations
that more closely examine the relationship between PCOT
practice and the sO level.
OTHER LIMITATIONS OF THE PRESENT STUDYExamining the changes brought about by thepractice of prayer, in general, or of Christian prayer, inparticular, involves some specific difficulties. First of all,there are several types of prayers (of request, of gratitude
or, among the most frequent ones in the studies of the last
decades, intercessory prayer), expressed in various forms,
individually, by reading a consecrated text, or
spontaneously, through free expression, or communally,
40

within a ritual. An inventory proposed by the psychology ofreligion analyzing the expressions and forms of prayer thatwould be relevant here identifies no less than 21 types ofprayers [80]. Problematic aspects also intervene in the
epistemological clarification of the general framework:
rigorous definitions of the concepts "religion" and"spirituality" [81], the empirical procedures appropriate to
the S/RE study or the place of divine intervention in
healing [82].
We have already seen that the measurement of
the dependent variables and the preliminary data of thepresent study contain many limitations arising from theexperimental design. The collection was done only once aday, for each participant separately, pre- and post-testing,for approximately one hour after waking up, after having
gone to the location of the laboratory.
Other limitations of the present study that can be
mentioned are those commonly found in studies using
salivary biomarkers, their values depending on age [83]and gender [84], but also on various aspects, such as diet[85], sleep quality [86] or the circadian rhythm [87]. On theother hand, as we have seen, stress, mood or mental
states influence the levels of salivary Cortisol, SIgA [88]and sAA [89], aspects that should not be neglected evenin the study of the changes brought about by the practiceof PCOT, the decrease in perceived stress being animportant indicator in evaluating the S/RE impact.
Other problematic aspects are related to thevalidity of the interpretations, formulated on the basis of
the data obtained regarding some of the used biomarkers.
For example, the correlations between salivary
testosterone levels and emotional reactions are still
unclear. For example, two studies contradict each other inthis respect, one suggesting that testosterone
administration (applied to a group of 16 women) caused adecrease in cognitive empathy [90], while the other, amore recent one, does not support a connection between
testosterone administration and decreased empathy [91].
On the other hand, there are also issues related to
the relevance of the levels of these salivary biomarkers
compared to their endogenous level. In this sense, thedebate regarding the possible inferences, regarding thelevels of endogenous oxytocin, measured in biologicalfluids, and the level of oxytocin released in the brain [92],is relevant, while it is worthwhile highlighting the fact thatthe insufficiency of evidence to support that endogenous
oxytocin levels in saliva and plasma, at rest, are stableenough for a valid assessment [93]. Finally, other studies
have suggested that oxytocin levels differ depending on
the method of measurement [94].
Fiziologia - Physiology • 2023.1(104)CONCLUSIONS
Hypothesis 3.1 - PCOT 30 min./ day, for 8 weeks, causes
a decrease in the levels of salivary Alpha Amylase (sAA);
Hypothesis 3.2 - PCOT 30 min./ day, for 8 weeks, causes
an increase in the levels of salivary Immunoglobulin A(SIgA);
Results: Statistical analysis of preliminary data did not
validate these hypotheses.
Hypothesis 3.3 - PCOT 30 min./ day, for 8 weeks, causes
a decrease in the levels of salivary Cortisol (sC) in thesubjects of the Study Group;
Result: We obtained statistically significant resultsthat validate hypothesis 3.3. However, further studies areneeded to distinguish the changes induced by theexperimental task, since the comparative analysis of SG
dynamics in relation to pre- and post-testing CG did notreveal significant differences.
Hypothesis 3.4 - PCOT 30 min./ day, for 8 weeks, causes
a decrease in salivary Testosterone (sT) levels;
Hypothesis 3.5 - PCOT 30 min./ day, for 8 weeks, causes
an increase in salivary Oxytocin (sO) levels.
Hypothesis 3.7 - PCOT 30 min./ day, for 8 weeks, changes
the levels of salivary interleukin 8 (sal IL-8);
Results: Statistical analysis of preliminary data did not
validate these hypotheses.
Hypothesis 3.6 - PCOT 30 min./ day, for 8 weeks, changes
the levels of Interleukin 6 (sal IL-6);
Result: Statistical analysis of preliminary data
validated this hypothesis. However, further studies areneeded to clarify whether PCOT always increases thelevel of IL-6, in the context of the other results mentioned,
which differed, some reporting an increase in IL-6
following an S/RE intervention, others showing a decreasein IL-6 levels.
Regarding the subsequent hypotheses, there
were no statistically significant differences between the

medians measured for SG and for CG, pre- and post-test,
for any of the seven variables: sAA, SIgA, sC, Salivary
Testosterone, Salivary Oxytocin (sO), Interleukin 6 (sal IL6) and Interleukin 8 (sal IL-8). These results correlate withthe poor differentiation between CG and SG, pre- andpost-testing, also found in two other related studies on
PCOT, regarding psycho-emotional changes and EEG
activity in the alpha, beta and gamma bands (results
pending publication).
For the study of changes induced by prayer, or
S/RE, fundamental epistemological clarifications areneeded, which concern the relationship between scienceand religion in general, and the manner in which thedemands of scientific study can be reconciled with thespecifics of S/RE. In particular, studying the physiologicalimpact of PCOT, measuring changes in salivary markers,
requires longer experimental intervals (allowing SG
individuals to better adapt to the cognitive-emotional,
behavioral and spiritual demands of PCOT) and more
complex collection protocols to include several moments,
immediately before and after the individual experience ofthe practitioners, and several times per day (forevaluations, in the case of salivary markers that register ahigh variability throughout the day).
The use of salivary markers to highlight somephysiological changes provides many advantages, but it
can also introduce many limitations. Therefore, to increase
the methodological quality of future studies on S/RE in
general and PCOT in particular, cross-assessments(psycho-emotional, electro-physiological and imaging
components) are needed, to obtain more consistent
packages of primary data, which allows for the comparisonof results regarding salivary biomarkers with those
provided by EEG recordings, imaging or various
psychometric scales.

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POSIBILE MODIFICĂRI FIZIOLOGICE INDUSE DE PRACTICA RUGĂCIUNII ÎN TRADIȚIA CREȘTIN-ORTODOXĂREZUMATUrmărim în studiul de față posibile modificări fiziologice induse de practica rugăciunii (în Tradiția Creștin Ortodoxă) - RTCO, după o practică
individuală de 30 de minute zilnic, timp de 8 săptămâni. Participanții (n=33) au fost împărțiți în două grupuri (Grup de studiu, n=16, cu media
de vârstă de 39,2 ani și Grup de control n=17, cu media de vârstă 38,6 ani). Subiecții Grupului de studiu – credincioși creștin ortodocși fărăpractica rugăciunii zilnice fiind instruiți pe parcursul unei sesiuni de 3 ore cu privire la specificul psiho-emoțional, comportamental și spiritual
al RTCO. Asupra subiecților din Grupul de control nu s-a desfășurat nicio intervenție. Înainte și după perioada celor 8 săptămâni de practică
zilnică a RTCO, 30 min, s-au colectat mostre de salivă, dimineața, la o oră de la momentul trezirii, fiind analizați comparativ, pre și posttestare nivelurile următorilor markeri salivari: Cortizolul, Alfa-amilaza salivară (AAs), Imunoglobulina A (IgA), Oxitocina, Testosteronul și
citokinele interleukina 6 (IL-6) și interleukina 8 (IL-8). Rezultatele obținute indică o scădere a Cortizolului salivar, semnificativă statistic și ocreștere a IL-6 la subiecții din Grupul de studiu, post-testare în raport cu nivelurile măsurate la momentul pre-testării, sugerând că RTCO arputea ameliora răspunsul fiziologic la stres și răspunsul inflamator. La ceilalți markeri salivari nu s-au obținut rezultate semnificative statistic.
Discutăm rezultate în contextul unor studii asemănătoare, evidențiind câteva limitări specifice studiilor care evaluează modificările fiziologice
antrenate de experiențele cu caracter spiritual/religios, în special a celor care utilizează markeri salivari, formulând și câteva recomandări
pentru creșterea calității metodologice a viitoarelor studii.
Cuvinte cheie: Rugăciunea creștin ortodoxă, modificări fiziologice, limitări ale studiilor care utilizează markeri salivari, Cortizolul salivar,
Interleukina 6 (IL-6), impactul rugăciunii zilnice, Alfa-amilaza salivară (AAs), Imunoglobulina A (IgA), Oxitocina, Testosteronul, interleukina 8 (IL8).

46

Fiziologia - Physiology • 2023.1(104)PRECISION DIAGNOSTIC METHODS IN SOLID
TUMORS – NEXT GENERATION SEQUENCING (NGS)HARICH OCTAVIA-OANA1, ANGHEL SIMONA1,2, TATU CARMEN1,2, TANASIE GABRIELA1,2,PĂUNESCU VIRGIL1,2, PANAITESCU CARMEN1,2
1Department of Functional Sciences, Immuno-Physiology and Biotechnologies Center (CIFBIOTECH), „Victor Babeș” Universityof Medicine and Pharmacy Timisoara
2Centre for Gene and Cellular Therapies in the Treatment of Cancer, Clinical Emergency Hospital „Pius Brinzeu” Timisoara

ABSTRACT
Cancer is an increasingly health problem worldwide, and new diagnostic methods are developing each year. Next generation sequencing (NGS)
represents already a state-of-the-art method in this field, while mutation-based therapies are standard-of-care in many countries for cancer
treatment. We present the methods and results that can be obtained using an NGS platform from IonTorrent, while employing a limited panel ofgenes (HotSpot Panel) and appropriate software for results analysis. This method is very fast (2-3 days) and requires low amout of DNA as input
material. The analysis reports using either IonReporter or Oncomine Reporter software can give the clinicians information related to mutations
localisation, type of mutations (synonymus, missense), mutation-targeted therapies and ongoing clinical trials, which could be of real bennefit forthe oncologic patients.
Key words: NGS, mutation-based therapy, solid tumors

INTRODUCTION
Worldwide, in 2020, there were approximately 16.1
million new cases of cancer in the 20-79 age group, roughly
equally divided between the two sexes: approximately 8.4
million new cases in men and 7.7 million new cases inwomen. From the point of view of localization, the cases are
distributed as follows: 12.9% breast cancer, followed by lungcancer (11.5%), colorectal (9.7%), prostate (7.4%), stomach(5.6%), liver (4.9%) and cervix (3.6%) (Figure 1) [1].

cancers were recorded in close connection with the following
locations: breast, colorectal, cervix, lung, uterine body, ovary,thyroid (Figure 2) [1].

Fig. 2. Cancer incidence in Romania, in 2020, in the
20-79 age group.

Fig. 1. Worldwide cancer incidence in the year 2020, in
the age group 20-79 years.
In Romania, in 2020, in the 20-79 age group, 84 245 newcases of cancer were registered, with a significantly higher
number of cases in males, namely 46,675 cases, compared
to 37,570 cases in the female sex. In the male sex, the mostfrequent cancers were recorded in close connection with thefollowing locations: lung, prostate, colorectal, urinary bladder,stomach, liver, larynx. In the female sex, the most frequent

The present study proposes the identification anddevelopment of modern methods of diagnosis andtherapeutic orientation in cancer (solid tumors) – nextgeneration sequencing (NGS). The aim of the study was todevelop a structured strategy for molecular diagnosis incancer, based on the evaluation of critical parameters, which
will help oncologists in choosing the most effective
therapeutic decision for patients with cancer, especially inthe case of solid tumors. The study will also generate
methods for precision molecular diagnosis and facilitate
access to clinical trial information for cancer patients.
From the point of view of the expected research results,
related to the methods used, it will be possible to establish:
47

Fiziologia - Physiology • 2023.1(104)a precision molecular diagnosis in oncological conditions; atherapeutic indication based on gene mutations - anti-tumor
therapies with potential benefit; information about genes
with mutations (COSMIC, JaxCKB, CiVIC) and theirinvolvement in the process of tumorigenesis; potential
neoepitopes targetable by specific immunotherapies.
The procedures used in this study were performed onparaffin blocks containing tumor tissue, which were
processed for nucleic acid (DNA) purification and next
generation gene sequencing (NGS).
MATERIALS AND METHODS
Samples: paraffin-embedded tumor tissue
DNA extraction kits: GeneJET FFPE DNA Purification Kit
DNA measurement kit: Qubit dsDNA HS Assay
Analyzed genes panel: Hotspot Panel – 49 genes and207 hotspots
Analysis device: Personal Genome Machine (PGM) Ion
Torrent, Ion Gene Studio S5
NGS
datainterpretation
software:Ion
Reporter/Oncomine Reporter.
1. Purification of DNA from paraffin-embedded
tumor tissue samples
Formalin fixed and paraffin embedded (FFPE) tissue
samples were obtained from surgical resection or biopsy
samples. Each procedure can yield up to 8 μg of genomic
DNA, which can be eluted in 20-80 μL of elution buffer. Highquality eluted DNA can be used directly for further
applications such as qPCR, PCR, NGS library preparation,or stored at -20 °C.
1. Add 200 μL of digestion buffer to a microcentrifuge
tube containing one or more sections (up to 8) of FFPE
tissue. Incubate for 3 minutes at 90 °C, mixing occasionally;
after incubation, mix vigorously with a vortex mixer tocompletely dissolve the paraffin. The samples are cooled toroom temperature.
2. Add 20 μL of proteinase K solution and mix vigorously
by vortexing or pipetting to obtain a uniform suspension. The
sample is incubated at 65 °C for 50 min in a thermoshaker
or water bath, vortexing occasionally (300-400 rpm).
3. Transfer the samples to the thermoblock set at atemperature of 90 °C and incubate for 40 min.
4. Centrifuge hot samples at 6000 × g for 1 min andtransfer 200 μL of digested lysate to a new 1.5 mL
microcentrifuge tube.
5. Add 10 μl of RNase A solution and mix vigorously byvortexing, then leave at room temperature for 10 minutes.
6. Add 200 μl of binding buffer.
7. Add 400 μl of ethanol (96-100%) to the sample tube.
8. Transfer the lysate to GeneJET DNA Purification
Columns, which are inserted into the collection tube, andcentrifuge for 1 minute at 6000 × g.
9. Wash 2x with 500 μl of wash buffer solution 1 and 500
μl of wash buffer solution 2, centrifuge for 3 minutes at
maximum speed (≥ 12,000 × g).

48

10. Samples are eluted in 50 μl of elution buffer.
After DNA purification, to determine the concentration of
nucleic acids, we used the QubitTM 3.0 Fluorometer
together with the QubitTM dsDNA HS Assay Kit (Invitrogen,
Life Technologies)(Figure 3).

Fig. 3. Steps of DNA concentration determination.
Using the QubitTM 3.0 Fluorometer and high-sensitivity
DNA concentration reagents, the concentration of doublestranded DNA is measured for further investigation.
(adapted from the manufacturer's procedures manual [2]1. Prepare 2 tubes for reading the standards and one
tube for each sample;
2. Prepare the working solution (Qubit Working Solution)by diluting the Qubit reagent 1:200 in the Qubit buffer
solution; for each sample and standard prepare 200 µl ofworking solution;
3. Prepare the tubes for testing;
4. Vortex all tubes for 2-3 seconds;
5. Incubate the tubes at room temperature for 2 minutes;
6. Insert the tubes into the dedicated space of the Qubit
3 fluorimeter and read the concentrations.
2. Preparation of libraries
To prepare the libraries we used the Ion AmpliseqTM
Cancer Hotspot Panel v2 Chef-Ready kit. This kit combines
a single panel of primers with the necessary reagents to
prepare libraries of frequently mutated regions in humantumor genes, subsequently using the Ion Chef™ system.
The automated workflow uses the same chemistry as the
manual library construction workflow, providing equivalent or
better performance with exceptional reproducibility, ontarget reads and sample balance. Automated processing
and library generation using the Ion Chef system reduces
actual manual work time to less than 15 minutes.
The Ion AmpliseqTM Cancer Hotspot Panel v2 ChefReady Kit contains two components:-

Ion AmpliseqTM Cancer Hotspot Research Panel
Primers;
Kit Ion AmpliseqTM for Chef DL8.

The workflow is simple from sample to data acquisitionand can be completed in less than two days, offering the

Fiziologia - Physiology • 2023.1(104)following features and benefits:---

Producing high quality DNA libraries using the Ion Chef
system;Proven performance of Ion Ampliseq™ technology,
FFPE compatibility, uniform coverage for efficientsequencing, accurate variant calling and variant
detection enhanced by Torrent Suite™ software;
Ability to scale and multiplex on Ion™ chips based ondesired throughput needs, leading to cost savings andefficiency;
Simplified workflow and reduced work time when usedwith the Ion Ampliseq™ Direct FFPE DNA kit;
Low input amount of only 1 ng per sample;

The Ion AmpliSeq Cancer Hotspot Panel v2 is designed
to amplify 207 amplicons covering approximately 2,800
COSMIC mutations for 50 oncogenes and tumor suppressor
genes. For gene sequencing, this panel must be used with
the Ion AmpliSeq™ Library Kit 2.0.
3. Gene sequencing using the IonTorrent platform
The sequencing procedures of the sample libraries
created in the previous step are performed in the automated
NGS system that includes both IonChef and IonS5 Gene
Studio, comprising the following steps:
1.

2.

3.
4.

Planning a sequencing/reading (Planned Run) – it isnecessary both for loading the chips and for the actual
reading;
Loading the chips with the amplicon libraries resulting
from the previous steps, which is done in the automatic
IonChef system;
Preparation of the gene sequencer Ion GeneStudio S5;
Sequencing itself – reading the chips;

Solutions, reagents, and consumables are used to load
the chips, and this procedure is performed sequentially foreach chip, depending on the size of the chip and the numberof libraries recommended for each chip.
In this study we used Ion 530TM chips (ThermoFisher
Scientific), which can be loaded with 4-8 libraries preparedwith the Cancer HotSpot panel. In the case of this study, theIon 530 chips were loaded with 8 libraries each (derived from
8 individual samples).
The chip loading procedure was performed
automatically in the IonChef system, requiring the following
solutions, reagents and consumables: Ion S5TM Chef
Supplies, Ion S5TM Chef Solutions, Ion 510TM, 520TM &530TM Chef Reagents (ThermoFisher Scientific).
A volume of 25 µl was pipetted from each amplicon
library (containing 8 samples) into the dedicated tube of Ion
510TM, 520TM & 530TM Chef Reagents. Consumables,
solutions and reagents were positioned according to Figure
4.

Fig. 4. Positioning the components inside the
IonChef for chip loading. 1. Empty support for spikes; 2.
Foil for sealing; 3. Tip box; 4. PCR plate; 5. Ion 510TM,
520TM & 530TM Chef Reagents cartridge; 6. Solution
cartridge; 7. Centrifuge tubes; 8. Library enrichment
cartridge; 9. Chip-adapter assembly [2].
At the end of the automatic chip loading (templating)procedure, 2 chips will result, each of which has the 8
libraries generated in the previous step.
The gene sequencing procedure must be synchronizedwith the loading of the chips and must be initiated within 2hours of obtaining them because the quality of the libraries
is ensured for an interval of 4-6 hours, under conditions ofkeeping the chips in a refrigerator , at a temperature of 4-8o
C.
Sequencing planning and the chip sequencing program
was previously performed taking into account the following
steps and the type of samples/libraries loaded onto the chips:---

Select the AmpliSeq DNA program;
Select Ion GeneStudio S5 System as a tool from theprogram menu;
Select the type of kit used for library preparation AmpliSeq Cancer Hotspot Panel v2;
Select the type of barcodes used – Ion Xpress;
Select the kit used to load the chips - Ion 510TM, 520TM& 530TM Chef;
Select the Ion S5 Sequencing Kit – used for genesequencing;
The right number of flows is chosen, appropriate to thesize of the amplicons loaded on the chips - in our casethe amplicons have a size of approximately 200 basepairs, so 500 flows are needed for the sequencing
procedure;
The reference sequence of the genome is chosen, withwhich the sequences from the libraries will be comparedto identify mutations – hg19 (Homo sapiens);
Target regions and hotspot regions are selected Cancer Hotspot Panel v2;

49
Fiziologia - Physiology • 2023.1(104)--Enter the number of barcodes used for each chip – 8
samples/8 libraries/8 barcodes, noted starting from
IonCode_0101 (CTAAGGTAAC);
Select the programs in which the generated data can beimported for analysis – IonReporter; the generated .vcf
files can be later analyzed using the Oncomine Reporter
software.

The Ion GeneStudio S5 system is initialized using
specific reagents, contained in the Ion S5TM Sequencing
Reagents cartridge, a used chip, washing solutions (Ion
S5TM Wash Solution) and cleaning solutions (Ion S5TM
Cleaning Solution), positioned inside the device. The
solution of wash is sufficient for a single instrument
initialization (sequencing 2 chips, providing 1200 flows, 500
flows/chip sequenced being required), while the cleaning
solution can be used for 4 initializations of the Ion
GeneStudio S5 system, cleaning the device after
sequencing 2 chips.
Sequencing data acquisition was performed on Torrent
Server, Torrent_Suite version 5.12.3.
RESULTSNGS quality report
Gene sequencing quality ratio refers to a series ofparameters that must fall within certain limits for thesequencing procedure to be declared correctly performed.
Figure 5 shows the following parameters: the degree ofloading and coverage of the sequenced chip (ISP Loading
and ISP Density), which are met, being 93%; the total
number of reads and the useful reads performed within thegene sequencing (36%) - fulfilled; polyclonality must be < 50%(47% in the example below); the length of the reads must fallwithin the amplicon size limits (for amplicons generatedusing the Cancer HotSpot panel, the amplicon size is < 200bp).

Fig. 6. Qualitative parameters of NGS. The informationrefers to the percentage of aligned bases, the average sizeof the analyzed fragments and the degree of (coverage).
One of the analysis and interpretation software used forthe data obtained at NGS was IonReporter Software. This
program provides us with information regarding thechromosomal location of the sequenced gene, the type ofmutation (SNV-type mutations were selected - missense,
frameshift/non-frameshift deletions and insertions,nonsense), the gene at which the mutation occurred, themutation at nucleotides, amino acid substitution modification(in the case of missense mutations), as well as the targeted
therapeutic indication for the identified mutation (Figure 7).

Fig. 7. Example of data analysis using the IonReporter
software.
Fig. 5. Gene sequencing quality report. The
characteristic parameters of chip loading, useful sequences
resulting from repeated reads and the size of these reads
are represented.
The quality report also includes information related tothe alignment of the bases in the analyzed sequences, thetotal number of bases, the average size of the amplicons, aswell as the degree of coverage (coverage), the importantparameters of NGS (Figure 6).

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The Oncomine Reporter analysis program is a complex
software that identifies, based on gene mutations, clinicallysignificant biomarkers for which targeted therapies exist in
the specified cancer type or in other cancer types approvedby oncology authorities: FDA, ESMO, EMA, NCCN. The
relevant therapies, monotherapy or combined therapy, for
the identified mutations are also specified, as well as thecontraindications of immunotherapies in the case of certain
gene mutations. In the Variant details section, the previously
mentioned parameters of the IonReporter software aredescribed, in addition, there is the possibility to quantify theallelic frequency of the mutant variant, which is an extremely

Fiziologia - Physiology • 2023.1(104)important parameter in differentiating between somatic andinherited mutations (germline), according to studies
conducted by Coffe et al., 2017 [3]. An important chapter in
the analysis report obtained using the Oncomine Reporter
program is the identification and listing of existing clinical
trials targeting the identified mutations/sample/patient(Figure 8).

Fig. 8. Example of data analysis using Oncomine
Reporter software
DISCUSSIONS
Tumor sequencing has revealed new genomic
aberrations that are responsible for tumor growth andprogression, and these driver mutations can be clinicallyinhibited using targeted molecular therapies. Precisiononcology entered a new era nearly two decades ago with thedevelopment and approval of two targeted therapies:
trastuzumab, for the treatment of HER2-amplified metastatic
breast cancer, and imatinib, for the treatment of chronic
myeloid leukemia (CML) with fusion BCR-ABL [4,5].
Currently, the FDA has approved genotype-targeted
therapies for various tumor types. Inhibitors of EGFR, ALK
and ROS1 are routinely administered to patients with nonsmall cell lung cancer who have genomic alterations in oneof these genes [6,7]. BRAF mutations, which occur inapproximately half of patients with cutaneous melanoma,
can be targeted with multiple inhibitors of the MAPK
signaling pathway [8,9]. Furthermore, in metastatic
colorectal cancer, EGFR-targeted therapies such as
cetuximab and panitumumab are specifically administeredto patients without mutations in the KRAS and NRAS
oncogenes [10]. By creating databases of genes and theiralterations, present in tumors with different anatomical
locations, the centralization of data from clinical trials canhave a higher level of precision, thus making the testing andapproval of new therapies more efficient.
The diversity of clinically relevant mutations means thatmolecular profiling platforms must be comprehensive andflexible to incorporate all classes of genomic alterations.
Hotspot panels are widely used to detect mutations in EGFR,
BRAF and/or other genes.
Molecular profiling of tumor samples has been
increasingly used in a wide range of cancer types, andresults have reaffirmed the ubiquity of specific driver
mutations in many different histologically defined tumor
types [11,12]. BRAFV600E mutations, which havehistorically been associated with melanoma, are found at
significant frequencies in patients with NSCLC, colon cancer,
thyroid cancer, multiple myeloma, glioma, or pancreaticcancer. HER2 amplifications, once thought to be exclusive
to HER2-positive breast cancers, are now consideredrelatively common in patients with esophageal cancer,
breast cancer, bladder cancer, or endometrial cancer. These
alterations can all be targeted therapeutically, and therefore
extensive profiling of the mutations, regardless of lineage, isneeded to ensure that both patients with common cancers
and those with rare cancers have the opportunity to receive
the treatment optimally available. Drugs that have received
FDA approval for use in a specific tumor type, such as the
BRAF inhibitors vemurafenib and dabrafenib in melanoma
patients, can be prescribed off-label for patients withdifferent types of cancer that share the same target lesions.
The emergence of identical or similar alterations that canbe targeted in multiple cancer types has also prompted thedevelopment of new clinical trial designs to systematicallytest the efficacy of drugs in different lines. In one such study
design, so-called basket trials, patient eligibility is defined
based on the presence of a specific genetic alteration rather
than a specific tumor type [13]. These trials involve targeted
medication and are suitable for mutations that occur with low
frequency in many cancers, where individual diseasespecific trials are unlikely to accrue enough patients toprovide a statistically significant assessment of treatment
response. Such trials have emerged as an effective way to
expand access to specific targeted therapies for patients
who have the appropriate alterations, particularly in patientswith rare cancers for which clinical trials have not been
widely available. The results of several such studies havealso revealed the importance of tumor type in conditioning
the response to targeted therapies [14,15]. These trials arein contrast to "umbrella" trials, which involve multiple cohorts
and are designed to test different targeted agents in patientswith different mutations in a single cancer type, where cohort
allocation is determined based on molecular profiling [16,17].
The US National Cancer Institute (NCI) has run an ambitious
trial, the Molecular Analysis for Therapy Choice (NCIMATCH) initiative, which functions as an umbrella basket oftrials, involving centralized molecular profiling using astandardized panel of hotspots in participating diagnostic
laboratories to assign patients to one of >30 treatment arms.
Thus, the use of NGS methodologies for identification of
mutations in cancer could be of great benefit for the
oncologic patients, but also for the research purposes withthe aim of finding new targets on tumor cells and tumor
microenvironment. This could be one step forward to
precision medicine and personalized treatment in cancer.

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METODE DE DIAGNOSTIC DE PRECIZIE ÎN TUMORILE SOLIDE – SECVENȚIEREA DE GENERAȚIE URMĂTOARE (NGS)REZUMAT
Cancerul este o problemă de sănătate din ce în ce mai mare la nivel mondial, iar noi metode de diagnostic se dezvoltă în fiecare an. Secvențierea
de generație următoare (NGS) reprezintă deja o metodă de ultimă generație în acest domeniu, în timp ce terapiile bazate pe mutații sunt standardde îngrijire în multe țări pentru tratamentul cancerului. Prezentăm metodele și rezultatele care pot fi obținute folosind o platformă NGS de la
IonTorrent, utilizând în același timp un panou limitat de gene (HotSpot Panel) și software adecvat pentru analiza rezultatelor. Această metodă
este foarte rapidă (2-3 zile) și necesită o cantitate mică de ADN ca material de intrare. Rapoartele de analiză care utilizează software-ul
IonReporter sau Oncomine Reporter pot oferi clinicienilor informații legate de localizarea mutațiilor, tipul de mutații (sinonim, missense), terapii
vizate de mutații și studii clinice în curs, care ar putea fi de real beneficii pentru pacienții oncologici.
Cuvinte cheie: NGS, terapie bazată pe mutații, tumori solide

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