TUBB8 mutations as a cause of oocyte maturation abnormalities: presentation of oocyte and embryo profiles and novel mutations.

RESEARCH QUESTION: What are the embryonic profiles and oocyte maturation dynamics in patients with tubulin beta eight class VIII (TUBB8) mutations leading to oocyte maturation abnormalities (OMAS), and are pregnancies possible in this population? DESIGN: A prospective cohort study was undertaken in a private fertility clinic between January 2019 and December 2022. Whole-exome genomic studies (WES) were performed to detect mutation types. In-vitro maturation (IVM) was compared in 18 subjects: nine with TUBB8 mutations, and nine without TUBB8 mutations to act as the control group. The distributions of oocyte maturation and embryonic development profiles were recorded. IVF and IVM outcomes of the 18 cases were evaluated. The primary outcomes were the embryonic profiles and maturation dynamics of oocytes derived from IVF or IVM in women as related to TUBB8 mutations. RESULTS: Mutations were detected in 52 of 89 (58.4%) women who underwent WES analysis. Twelve TUBB8 mutations were detected in nine women (10.1%) with OMAS. Seven novel TUBB8 mutations were noted. Two pregnancies were obtained in women with c.535 G>A TUBB8 mutations. When comparing IVM outcomes between women with and without TUBB8 mutations, there were no differences in oocyte, embryo or pregnancy parameters (P>0.05 in all cases). CONCLUSIONS: It is clear that further TUBB8 mutations which cause oocyte or embryonic arrest will be detected in future. Although biochemical or ectopic pregnancies may be possible in some of these women, no live births or ongoing pregnancies have been reported to date.

A novel SLC35D1 variant causing milder phenotype of Schneckenbecken dysplasia in a large pedigree.

SLC35D1 gene encodes UDP-glucuronic acid/UDP-n-acetylgalactosamine dual transporter protein and transports organic or inorganic molecules across cellular membranes. SLC35D1 gene pathogenic variants causes Schneckenbecken dysplasia (SHNKND) which is a rare lethal autosomal recessive disorder characterized by the snail-like pelvis, flattening of vertebral bodies, short and broad long bones with a dumbbell-like appearance, thoracic hypoplasia. Only six cases with homozygous SLC35D1 variants have been reported to date, and all of these cases were lost in the perinatal period. Here we report different family members with a novel SLC35D1 variant who presented a milder phenotype of SHNKND. The affected patients have common clinical features such as short stature, mild mesomelia, shortening of the lower extremity, genu valgum, and narrow thorax. Exome sequencing of the proband revealed a homozygous missense variant of SLC35D1 gene, c.401 T > C (p. Met134Thr). The affected siblings, their two cousins, and their paternal uncle with a similar phenotype were also homozygous for the variant. This is the first case report of a family with a novel likely pathogenic variant (p. Met134Thr) and mild phenotypic features. It has the largest family with different ages of patients (ages ranged 4-31 years old) reported to date. The present report supports the evidence that the p. Met134Thr variant is responsible for a milder phenotype than previously reported cases with SLC35D1 pathogenic variants.

Early-infantile onset epilepsy and developmental delay caused by bi-allelic GAD1 variants.

Gamma-aminobutyric acid (GABA) and glutamate are the most abundant amino acid neurotransmitters in the brain. GABA, an inhibitory neurotransmitter, is synthesized by glutamic acid decarboxylase (GAD). Its predominant isoform GAD67, contributes up to ∼90% of base-level GABA in the CNS, and is encoded by the GAD1 gene. Disruption of GAD1 results in an imbalance of inhibitory and excitatory neurotransmitters, and as Gad1-/- mice die neonatally of severe cleft palate, it has not been possible to determine any potential neurological dysfunction. Furthermore, little is known about the consequence of GAD1 disruption in humans. Here we present six affected individuals from six unrelated families, carrying bi-allelic GAD1 variants, presenting with developmental and epileptic encephalopathy, characterized by early-infantile onset epilepsy and hypotonia with additional variable non-CNS manifestations such as skeletal abnormalities, dysmorphic features and cleft palate. Our findings highlight an important role for GAD1 in seizure induction, neuronal and extraneuronal development, and introduce GAD1 as a new gene associated with developmental and epileptic encephalopathy.

Demographic distributions and clinical results of assisted reproduction techniques in Turkey in 2019: a descriptive survey.

Objective: The aim of this study was to describe characteristics and outcomes of assisted reproductive technology (ART) cycles performed in 2019 in Turkey. Material and Methods: One-hundred and sixty-five ART centers in Turkey were invited to submit data. The survey was sent to center directors via e-mail with anonymous links by Qualtrics™. The survey involved questions about their patient characteristics, clinical practices, and outcomes. Results: Forty-one (24.8%) centers responded to e-mails, and data gathered from 25 centers was included in the analyses. In 25 centers, 18,127 fresh or frozen transfers were carried out during the study period, of which 7796 (43.0%) were fresh and the rest were either frozen (45.2%) or embryo transfers (ET) with preimplantation genetic testing (PGT) (11.8%). The live birth rate per ET was as 30.6%, 40.1%, and 50.7% in fresh, frozen and PGT cycles, respectively. A single embryo was transferred in 65.3% of all transfers and singleton live births comprised 86.1% of all deliveries. For cycles with intrauterine insemination, 1407 were started in 2019, and 195 clinical pregnancies, 150 live births with 19 multiple pregnancies occurred. A total of 1513 ART cycles were initiated for foreign patients. Russia (29.6%), Germany (7.4%), Iraq (4.6%), Uzbekistan (3.1%), and Syria (1.4%) were the top five countries with most patients coming to Turkey for ART. Conclusion: The survey results are in parallel with the reports of international institutions and organizations. With repeated editions, the data collected with annual surveys can be used to inform ART practices in the coming years.

Detection of IL-1ß in culture media supernatants of pre-implantation human embryos; its relation with embryo grades and development.

In this study, we aimed to determine whether human embryos secrete interleukin-1ß (IL-1ß) into culture media and its correlation with embryo grade and development. Culture media supernatants of 100 embryos obtained from 39 cycles of 38 patients and cultivated individually were collected 2 and 3 days after intracytoplasmic sperm injection (ICSI). IL-1ß concentrations of samples were determined with ELISA and compared with embryo grades and blastomere numbers. Embryo grades and the amount of IL-1ß they secreted were found not to be correlated (p:0.559). Numbers of blastomeres each embryo had at 2nd and 3rd days were found to be correlated with IL-1ß secreted (p:0.00 and p:0.00, respectively). Mean amount of IL-1ß secreted by the embryos from ejaculated sperm cycles were found to be significantly higher than that of embryos from TESE cycles (p:0.016). Patient age and etiology of infertility were not correlated with the amount of IL-1ß secreted and embryo grade. In conclusion, preimplantation human embryos secrete IL-1ß in their media in amounts correlated with their blastomere numbers.

Prenatally diagnosed case of 22q11.2 deletion syndrome associated with pulmonary artery aneurysm.

Here, we report a new case with chromosome 22q11 deletion and cardiac anomaly diagnosed prenatally by echocardiography. Fluorescence in situ hybridization (FISH) analysis demonstrated a heterozygous deletion at 22q11.2. Echocardiography revealed ventricular septal defect, pulmonary atresia, and aneurysm of the main pulmonary artery and its branches. Pulmonary artery aneurysm (PAA) is rarely seen in patients with 22q11.2 deletion syndrome (22qDS). In this case, PAA was found by prenatal echocardiographic examination at the 25th week of gestation. To date, no prenatally diagnosed case of 22qDS with PAA has been reported. This is the first 22qDS case with PAA that was detected prenatally by FISH analysis.

Can PCNA and LIM15 gene expression levels predict sperm retrieval success in men with non-obstructive azoospermia?

INTRODUCTION AND OBJECTIVES: It is necessary to be able to predict sperm retrieval before microdissection testicular sperm extraction (mTESE) in azoospermic men. This study established the importance of proliferating cell nuclear antigen (PCNA) and LIM15 gene expression levels in predicting the success of sperm retrieval by mTESE. MATERIALS AND METHODS: One hundred and forty-three men who were diagnosed with non-obstructive azoospermia (NOA) were included in the study. Patients' age, total testosterone and follicle stimulating hormone values, testicular volume and testicular histology were recorded by prospectively. PCNA and LIM15 gene expression levels were determined by real-time PCR in the materials from both ejaculate and testicular specimens. RESULTS: Testis volume and histology were the most important factors in predicting the sperm retrieval rate (SRR). The PCNA and LIM15 gene expression levels measured in testicular tissues and the LIM15 gene expression levels measured in ejaculate significantly correlated with the SRR in mTESE (p=0.038, p=0.022, and p=0.004, respectively). Although the PCNA gene expression level measured in ejaculate was higher in men with successful sperm retrieval, the difference was not statistically significant (p=0.061). According to the multivariate logistic regression analysis, testicular volume and LIM15 gene expression level in ejaculate were independent predictive parameters for sperm retrieval. CONCLUSION: The data showed that LIM15 gene expression level in ejaculate is a useful molecular marker to predict the SRR before mTESE.

An Incidental Detection of a Cryptic Complex Chromosome Rearrangement Found During NGS Based PGT-SR: A Case Report.

Background: Complex chromosome rearrangements (CCRs) involve more than 2 chromosomal breakpoints and cause the exchanges of chromosomal segments between two or more chromosomes. The carriers of CCRs have normal phenotypes, but they have a higher risk of reproductive failure. Case Presentation: This paper presents a couple with a history of two affected children, one spontaneous abortion, three in vitro fertilization (IVF) failures, and one healthy boy who were referred to our laboratory for preimplantation genetic testing (PGT). The wife had been evaluated as a carrier of 46,XX,t (2;6)(p21;p25); therefore, four IVF treatment cycles supported with PGT for this translocation had been performed in different IVF centers until the couple consulted our laboratory. Only one of these four IVF attempts had resulted in a healthy boy and this IVF study had been performed with fluorescence in situ hybridization (FISH)-based preimplantation genetic testing for structural chromosomal rearrangements (PGT-SR). The fifth IVF study with next-generation sequencing (NGS)-based PGT was performed by our laboratory and no healthy embryo was found in evaluated 6 embryos. During our NGS-based PGT, the cryptic involvement of 12p was firstly detected. FISH with chromosome 2,6, and 12 specific probes revealed that the mother was a carrier of a balanced 3-way translocation of 46,XX,t(2;6;12)(p21;p25;p13). Conclusion: NGS based PGT-SR method is an accurate method for detecting the copy number variations and is helpful to find out the cryptic CCRs.

Oocyte maturation abnormalities - A systematic review of the evidence and mechanisms in a rare but difficult to manage fertility pheneomina.

A small proportion of infertile women experience repeated oocyte maturation abnormalities (OMAS). OMAS include degenerated and dysmorphic oocytes, empty follicle syndrome, oocyte maturation arrest (OMA), resistant ovary syndrome and maturation defects due to primary ovarian insufficiency. Genetic factors play an important role in OMAS but still need specifications. This review documents the spectrum of OMAS and to evaluate the multiple subtypes classified as OMAS. In this review, readers will be able to understand the oocyte maturation mechanism, gene expression and their regulation that lead to different subtypes of OMAs, and it will discuss the animal and human studies related to OMAS and lastly the treatment options for OMAs. Literature searches using PubMed, MEDLINE, Embase, National Institute for Health and Care Excellence were performed to identify articles written in English focusing on Oocyte Maturation Abnormalities by looking for the following relevant keywords. A search was made with the specified keywords and included books and documents, clinical trials, animal studies, human studies, meta-analysis, randomized controlled trials, reviews, systematic reviews and options written in english. The search detected 3,953 sources published from 1961 to 2021. After title and abstract screening for study type, duplicates and relevancy, 2,914 studies were excluded. The remaining 1,039 records were assessed for eligibility by full-text reading and 886 records were then excluded. Two hundred and twenty seven full-text articles and 0 book chapters from the database were selected for inclusion. Overall, 227 articles, one unpublished and one abstract paper were included in this final review. In this review study, OMAS were classified and extensively evaluatedand possible treatment options under the light of current information, present literature and ongoing studies. Either genetic studies or in vitro maturation studies that will be handled in the future will lead more informations to be reached and may make it possible to obtain pregnancies.

Expanding the phenotypic spectrum of lupus erythematosus in Aicardi-Goutières syndrome.

OBJECTIVE: Aicardi-Goutières syndrome (AGS) is an early-onset encephalopathy resembling congenital viral infection that is characterized by basal ganglia calcifications, loss of white matter, cerebrospinal fluid (CSF) lymphocytosis, and elevated interferon-alpha levels in the CSF. Studies have shown that AGS is an autosomal-recessive disease linked to mutations in 5 genes, encoding the 3'-repair DNA exonuclease 1 (TREX1), the 3 subunits of ribonuclease H2 (RNASEH2A-C), and sterile alpha motif domain and HD domain-containing protein 1 (SAMHD1). In this study we further characterized the phenotypic spectrum of this disease. METHODS: Clinical and laboratory data were obtained from 26 patients fulfilling the clinical diagnostic criteria for AGS. Genomic DNA was screened for mutations in all 5 AGS genes by direct sequencing, and sera were analyzed for autoantibodies. RESULTS: In 20 patients with AGS, 20 mutations, 12 of which were novel, were identified in all 5 AGS genes. Clinical and laboratory investigations revealed a high prevalence of features (some not previously described in patients with AGS) that are commonly seen in patients with systemic lupus erythematosus (SLE), such as thrombocytopenia, leukocytopenia, antinuclear antibodies, erythematous lesions, oral ulcers, and arthritis, which were observed in 12 (60%) of 20 patients with AGS. Moreover, the coexistence of AGS and SLE, was for the first time, demonstrated in 2 patients with molecularly proven AGS. CONCLUSION: These findings expand the phenotypic spectrum of lupus erythematosus in AGS and provide further insight into its disease mechanisms by showing that activation of the innate immune system as a result of inherited defects in nucleic acid metabolism could lead to systemic autoimmunity.

Evaluation of in vitro fertilization parameters and estrogen receptor alpha gene polymorphisms for women with unexplained infertility.

PURPOSE: Association of ESR1 gene PvuII, XbaI and (TA)n microsatellite polymorphisms and woman infertility was evaluated. METHODS: Infertile(n = 104) and fertile(n = 107) women were included in this study. We performed polymerase chain reaction-restriction fragment-length polymorphism analysis for detecting ESR1 polymorphisms. RESULT(S): PvuII and XbaI polymorphisms confered risk for infertility in a simple dominant manner in which a significant relationship was observed between infertile and control women. Infertile women had fewer(<18) short repeat alleles in promotor region. ESR1 genotypes were compared concerning maturation, fertilization, pregnancy rates and embryo quality. Although no difference was found in terms of pregnancy rates, maturation and fertilization rates were significantly smaller in pp and xx genotypes. Also, pp genotypes had significantly lower number of good quality embryos. Long TA repeat in promotor was found to be associated with low fertilization rate. CONCLUSION(S): Polymorphisms at the ESR1 gene are associated with infertility in this Turkish infertile women population.

Globozoospermia: Do Y-chromosome microdeletions play a role in this rare spermatogenic disorder?

OBJECTIVE: To explore the possible relationship between Y-chromosome microdeletions and a rare spermatogenic disorder, globozoospermia. STUDY DESIGN: Twelve patients with 100% globozoospermia were evaluated. Each man was questioned about his medical and surgical history and underwent a thorough andrologic examination. Plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone and prolactin levels were measured. Routine sperm analysis and morphology with electron microscopy were done. A set of 17 Y-specific sequence-tagged sites spanning the 3 AZF regions and also RBM1, ZFY, SRY, sY78, CDY, BPY2 and PRY were tested to detect the existence of Y-chromosome microdeletions by polymerase chain reaction. RESULTS: The mean age of the patients was 36 years (range, 27-42). No patient had abnormal blood concentrations of FSH, LH, testosterone or prolactin. Semen analysis revealed normal values for volume (2.2 mL) and concentration (32 x 10(6)/mL) but subnormal values for motility (37%) and progressive motility (24%). On light and electron microscopy, all the spermatozoa were round headed, with abnormal morphologic features. Patients had normal 46,XY karyotyping. No microdeletion of the Y chromosome was detected in any patient. CONCLUSION: Although this study did not find any Y-chromosome microdeletions in patients with globozoospermia, the exact genetic locus resulting in this pathology requires further study.

Mandibuloacral dysplasia and LMNA A529V mutation in Turkish patients with severe skeletal changes and absent breast development.

Mandibuloacral dysplasia (MAD) is an autosomal recessive disorder characterized by acroosteolysis (resorption of terminal phalanges), skin changes (hyperpigmentation), clavicular hypoplasia, craniofascial anomalies, a hook nose and prominent eyes, delayed closures of the cranial sutures, lipodystrophy, alopecia, and skeletal anomalies. MAD patients are classified according to lipodystrophy patterns: type A and type B. The vast majority of MAD cases are caused by LMNA gene mutations. MAD patients with type A lipodystrophy (MADA) have been reported to have LMNA R527H, A529V, or A529T mutations. In this report, we describe two MADA patients with progressive skeletal changes, absent breast development, and cataract in addition to the classical MAD phenotype. Both patients were found to be homozygous for the Ala529Val mutation of the LMNA gene. Our female patient is the oldest MADA patient (59 years old) who has ever been reported with the LMNA mutation and also the LMNA Ala529Val mutation. This study is the second report on MADA patients with a homozygous Ala529Val mutation.

Effects of pentoxifylline and platelet activating factor on sperm DNA damage.

OBJECTIVE: Pentoxifylline and platelet-activating factor (PAF) have been used to increase sperm motility in embryology laboratories. In the present study, we aimed to investigate whether these agents pose sperm DNA damage using DNA sperm chromatin dispersion (SCD) assay. STUDY DESIGN: Following application of pentoxifylline and PAF, sperm samples of 50 individuals with different sperm parameters were compared to baseline in terms of DNA damage using SCD assay. Furthermore, the relationship between DNA damage and sperm parameters in predicting DNA damage was assessed. RESULTS AND CONCLUSIONS: Significant increase in DNA damage was observed following application of PAF and pentoxifylline. Furthermore, DNA damage was significantly increased with application of pentoxifylline compared to PAF. Sperm motility was observed to be a statistically significant indicator in predicting alterations in DNA damage in baseline and subsequent to application of PAF and pentoxifylline independent of sperm concentration and morphology. Increased DNA damage was observed in both groups following application of pentoxifylline and PAF. Furthermore, the increase in DNA damage was higher in samples treated with pentoxifylline compared to samples treated with PAF. Thus, PAF seems to be more innocent in choosing viable sperm cells and in achieving sperm motility in the in vitro fertilization laboratory.

Enzymatic digestion plus mechanical searching improves testicular sperm retrieval in non-obstructive azoospermia cases.

BACKGROUND: In non-obstructive azoospermic patients (NOA) besides the mechanical treatment, vital spermatozoa from the tissue obtained from testes by biopsy can be enzymatically prepared. OBJECTIVE: To increase the sperm recovery success of testicular sperm extraction (TESE), suitable for ICSI. STUDY DESIGN: Prospective, clinical study. In 177 consecutive men who presented with clinical and laboratory data indicating NOA, tissue samples were obtained by microdissection TESE method. Initially, mature spermatozoa were searched for by mechanical extraction technique shredding the biopsy fractions. In cases with no spermatozoa was observed after maximum 30 min of initial searching under the inverted microscope, the procedure was then followed by enzymatic digestion using DNAse and collagenase type IV. In cases of at least a mature spermatozoon could be obtained properly, ICSI was performed. RESULTS: Of 177 cases with NOA, conventional mincing method extended with enzymatic treatment yielded successful sperm recovery for ICSI in 102 (57%). Overall in vitro tissue-processing time for patients with sperm recovery failure after 30 min of mechanical searching, was between 80 and 105 min (mean 96+/-9). Cleavage, embryo transfer and clinical pregnancy rates in mechanical plus enzymatic TESE patients were not significantly different from those of only mechanically TESE performed patients (p>0.05). CONCLUSION: Combination of conventional TESE and enzymatic digestion is an effective method to recover spermatozoa suitable for ICSI. The benefit of the mincing combined with enzyme to sperm retrieval for NOA is firstly to shorten the mechanical searching time, thus minimizing further cellular damage as well as exposure to external conditions, and secondly to reduce the number of cases with sperm recovery failures.

Oogonial Precursor Cell-Derived Autologous Mitochondria Injection to Improve Outcomes in Women With Multiple IVF Failures Due to Low Oocyte Quality: A Clinical Translation.

BACKGROUND: Mitochondrial dysfunction has been suggested as a major cause of age-induced decline in oocyte quality. In the past, donor oocyte cytoplasmic transfer showed some success but was abandoned due to the concerns with heteroplasmy. Recent studies indicated presence of oogonial precursor cells (OPCs) in the human ovary, which could be an autologous source of "healthy mitochondria." We sought to investigate the clinical efficacy of OPC-derived autologous mitochondrial injection (AMI) to improve oocyte quality in women with multiple in vitro fertilization (IVF) failures. METHODS: The OPCs were isolated from laparoscopically obtained ovarian cortical pieces by cell sorting using a monoclonal anti-vasa homolog (anti-DDX) antibody. They were then disrupted and mitochondria were isolated. Reconstituted mitochondria were injected into each oocyte during intracytoplasmic sperm injection. Paired comparisons were made between the first failed cycles and the post-AMI cycles. RESULTS: Of the 15 women undergoing ovarian stimulation, 2 were canceled and 3 decided to pool oocytes for later AMI. In remaining 10 (mean age 34.7 ± 4.1), AMI significantly improved fertilization rates (49.7 ± 31.3 vs 78.3 ± 18.9; P = .03) with a trend for better embryo grades (2.3 ± 0.3 vs 3.1 ± 0.7; P = .08). Four of 10 women conceived after single frozen embryo transfer and 3 after confirmation of diploidy via array comparative genomic hybridization (aCGH) (clinical pregnancy/embryo transfer = 4/10). CONCLUSION: These data show encouraging results for AMI in comparison to previous failed IVF cycles.

Recombinant chromosome with partial 14 q trisomy due to maternal pericentric inversion.

BACKGROUND: 14q duplications caused by parental pericentric inversion of chromosome 14 are rarely reported and no clear genotype-phenotype correlation has been determined yet. CASE PRESENTATION: Here we reported a 7 years old female patient with recombinant chromosome characterized by 14 q duplication and originated from maternal pericentric inversion of chromosome 14. Principal clinical findings of the child include developmental delay, microcephaly, hypertelorism, low set ears, clinodactyly of fifth fingers, hypotonia, telecanthus and cardiac malformation. CONCLUSIONS: Her final karyotype was 46,XX,rec(14)dup(14q)inv(14)(p11.2q24)mat,arr14q24.1-qter(64,800,000-108,350,000 bp)x3.

Increased frequency of sister-chromatid exchange and altered alkaline comet assay scores in superovulation cycles for unexplained infertility.

OBJECTIVE: Sister-chromatid exchange analysis (SCE) and alkaline comet assay measure and analyze DNA damage in mammalian cells. This study assesses whether ovulation induction agents increase DNA damage in the lymphocytes of infertile women undergoing superovulation. STUDY DESIGN: Prospective case control study in a university based hospital. Baseline and hCG day peripheral blood samples were withdrawn from 20 women with undergoing superovulation for unexplained infertility and baseline and luteinizing hormone (LH) peak day samples were also withdrawn from another 20 infertile women with unexplained infertility. RESULTS: There was increased SCE frequency and DNA damage determined by comet assay on the hCG day compared to the basal state. The SCE increase was correlated with the hCG day estradiol (E2) but not with ampoules of follicular stimulating hormone (FSH). The SCE frequency was also increased naturally ovulating women; however this was significantly less than that in FSH receiving women. CONCLUSION: Increased DNA damage may indicate for increased potential for malignancies after superovulation.

Assessment of cytogenetic aberrations and comet assay in colorectal adenocarcinomas.

UNLABELLED: AIMS, BACKGROUND AND STUDY DESIGN: Few studies have investigated the karyotypes of colorectal carcinomas with emphasis on the correlation between cytogenetic findings and clinicopathologic features. The aim of our study involving 20 colorectal adenocarcinomas was to determine their genomic alterations at the chromosomal level by correlating the cytogenetic findings with the extent of DNA damage and clinicopathologic parameters and to compare the results with those of healthy controls. RESULTS: Cytogenetic evaluation of patients and controls revealed 10 abnormal karyotypes in patients with adenocarcinomas located in the rectum, sigmoid and rectosigmoid regions. Four had numerical and six had structural abnormalities. CONCLUSIONS: Statistical analysis revealed a significant difference compared with controls (P <0.01). The karyotypes and the extent of DNA damage assessed by the comet assay were also significantly correlated with tumor stage (P <0.01) using the Kruskal-Wallis non-parametric test, while no statistical significance was observed in relation to patient age and smoking.