Lithuanian University of Health Sciences Faculty of Medicine Department of Genetics and Molecular Medicine

Lithuanian University of Health Sciences

Faculty of Medicine

Department of Genetics and Molecular Medicine

Khaled Sayah

AZF MICRODELETIONS AND SPERM DISORDERS IN MALE

INFERTILITY:

A SYSTEMIC REVIEW

AZF MIKRODELECIJOS IR SPERMOS SUTRIKIMAI VYRŲ

VAISINGUME:

SISTEMINĖ APŽVALGA

Medical Integrated Master’s Study Programme

Supervisor:

Virginija Ašmonienė

MD, PhD

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TABLE OF CONTENTS

ETHICS COMMITTEE APPROVAL ... 7

ABBREVIATIONS ... 8 TERMS ... 9 METHODOLOGY ... 13 Inclusion criteria ... 14 Exclusion criteria ... 14 Data extraction ... 14 Statistical analysis ... 15 Heterogeneity ... 15 Publication bias ... 15 RESULTS ... 16 Study characteristics ... 16 Meta-analysis ... 16 DISCUSSION ... 25 CONCLUSION ... 27 REFERENCES ... 28

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SUMMARY

INTRODUCTION: Male infertility is a disorder that is believed to contribute to more than 50% of all couple infertility cases, and directly responsible for 20-30% of all cases. Detectable genetic disorders are responsible for 15%-20%, whereas 30%-60% of cases do not receive a clear diagnosis and are considered idiopathic. One of the most assessed genetic disorders in idiopathic male infertility is AZF locus microdeletions. The AZF region of the long arm of the Y chromosome plays an essential role in male sexual determination, maturation and development. The main and only presentation in these idiopathic cases is sperm disorders that are tested for by performing sperm analyses. The most common sperm disorders are azoospermia and oligozoospermia. This locus is assessed using polymerase chain reaction testing for sequence-tagged sites, sites are known for their role and their absence usually indicates the presence of a microdeletion.

AIM: Deduce a risk relationship between AZF locus microdeletions, sperm disorders and male infertility as case and control populations

OBJECTIVES: Analyze selected studies in terms of assessed AZF subregion and geographical location, analyze difference between different AZF microdeletion incidences in geographical subregions, determine risk difference of AZF microdeletion between case and control groups and analyze distribution of sperm disorders with respect to AZF microdeletions.

RESULTS: This review assessed 13 articles that studies AZF microdeletions across 3 different sperm disorders in cases of idiopathic male infertility as case-control studies. The number of case subjects was much larger than the number of control subjects. The results were an almost 2-fold risk of suffering a microdeletion in the case group compared to that of the control group.

CONCLUSION: The determined risk of suffering male infertility in case group versus control group when suffering an AZF microdeletion (a, b, c, d and others) was approximately 2:1. Most studies stemmed from Eastern Asia, and majority of studies chose to assess AZFc subregion microdeletions exclusively. AZFc microdeletions were the most prevalent finding. AZFc microdeletions accounted for 88% of all case subjects with azoospermia, oligozoospermia and oligoasthenoteratozoospermia

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SANTRAUKA

ĮVADAS: Vyrų nevaisingumas yra sutrikimas, kuris, kaip manoma, prisideda prie daugiau nei 50% visų poros nevaisingumo atvejų ir tiesiogiai atsakingas už 20-30% visų atvejų. Aptinkami genetiniai sutrikimai yra atsakingi už 15%-20%, tuo tarpu 30%-60% atvejų nėra aiškios diagnozės ir jie yra laikomi

idiopatiniais. Vieni iš labiausiai pasitaikančių idiopatinių vyrų nevaisingumo genetinių sutrikimų yra AZF lokusų mikrodelecijos. Y chromosomos ilgojo peties AZF sritis atlieka esminį vaidmenį vyrų seksualinei determinacijai, brendimui ir vystymuisi. Pagrindinis ir vienintelis šių idiopatinių atvejų pateikimas yra spermos sutrikimai, kurie tiriami atliekant spermos tyrimus. Labiausiai paplitę spermos sutrikimai yra azoospermija ir oligozoospermija. Šis lokusas vertinamas, naudojant polimerazės grandininės reakcijos tyrimą nuosekliai pažymėtoms vietoms, kurios yra žinomos dėl savo vaidmens ir jų nebuvimas paprastai rodo mikrodelecijos buvimą.

TIKSLAS: Nustatyti rizikos santykį tarp AZF lokusų mikrodelecijų, spermos sutrikimų ir vyrų nevaisingumo kaip atvejį ir kontroliuoti populiaciją.

TIKSLAI: Išanalizuoti pasirinktus tyrimus, vertinant AZF subregioną ir geografinę padėtį, išanalizuoti skirtingų AZF mikrodelecijų paplitimo skirtumus geografiniuose subregionuose, nustatyti AZF mikrodelecijos rizikos skirtumą tarp atvejų ir kontrolinių grupių bei išanalizuoti spermos sutrikimų pasiskirstymą pagal AZF mikrodelecijas.

REZULTATAI: Šioje apžvalgoje buvo vertinama 13 straipsnių, kuriuose buvo nagrinėta 3 skirtingi spermos sutrikimų AZF mikrodelecijas idiopatinio vyrų nevaisingumo atvejai, atvejų kontrolės tyrimo būdu. Atvejo subjektų skaičius buvo daug didesnis nei kontrolinių subjektų skaičius. Rezultatai parodė, jog 1.86:1 atvejų grupėje rizikuoja nukentėti nuo mikrodelecijų, lyginant su kontroline grupe.

IŠVADA: Nustatyta rizika patirti vyriškus nevaisingumo atvejus atvejų grupėje, palyginti su kontroline grupe, patiriant AZF mikrodeleciją (a, b, c, d ir kita) buvo maždaug 2:1. Dauguma tyrimų buvo atlikta Rytų Azijoje, ir dauguma tyrimų pasirinko vertinti išimtinai tik AZFc subregiono mikrodeleciją. AZFc mikrodelecijos buvo labiausiai paplitusios. AZFc mikrodelecijos sudarė sudarė 88% visų atvejų su azoospermija, oligozoospermija ir oligoastenoteratozoospermija.

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ACKNOWLEDGEMENTS

Special thanks for professor Virginija Ašmonienė and doctor Marius Šukys for their patience and help with preparing this literature review.

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CONFLICT OF INTERESTS

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ETHICS COMMITTEE APPROVAL

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ABBREVIATIONS

PAR – pseudoautosomal regions

NRY – non-recombining region of Y chromosome MSY – male specific region of Y chromosome AZF – azoospermia factor

STS – sequence-tagged site PCR – polymerase chain reaction Azoosp. – azoospermia Oligozoosp. – oligozoospermia Oligoasthenoterato. – oligoasthenoteratozoospermia OR – Odds ration RR – Risk ratio CI - Confidence interval PI – Prediction intervals

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TERMS

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INTRODUCTION

Human infertility is a term clinically designated by the World Health Organization as a “disease of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse”. 1_{Male infertility, infertility only due to male causes, is believed to}

contribute to 50% of all infertility cases, and solely responsible in 20-30%.2Most common causes and associated factors known to be involved with male infertility are: congenital or acquired urogenital abnormalities, malignancies, urogenital tract infections, endocrine disturbances and disorders, systemic diseases, immunological diseases, disturbances of erection/ejaculation, and idiopathic infertility.3,7 Genetics are known to play a role in a wide variety of such disorders that can lead to male infertility, as well as being directly responsible in idiopathic cases. Identifiable genetic abnormalities are believed to contribute in 15%-20% of the most severe forms of male infertility, while 30%-60% remain without a clear diagnosis. Genetic causes are generally categorized as chromosomal disorders, Y chromosome microdeletions, mitochondrial DNA mutations, monogenic disorders and multifactorial disorders.

The Y chromosome is considered to be the most important factor when considering genetic male infertility when all other causes are excluded, specifically in idiopathic cases. It is responsible for male sexual determination, maturation and development. The Y chromosome is cytogenetically divided into: pseudo autosomal regions (PARs) which undergo crossing with the PARs of the X chromosome; and the non-recombining region (NRY) or the male specific region (MSY); consisting of the long arm (Yq) and the short arm (Yp), which does not. The euchromatic segment of the Yq (Yq11); genetically active unlike the heterochromatic one; has been the main focus of consideration in cases of male infertility presenting only with primary sperm disorders.

Based on observation of cytogenetically visible deletions, the first spermatogenesis-related factor to be proposed in this topic was the azoospermia factor (AZF) locus. The AZF gene locus has since been heavily studied in depth, and has been designated as recurrent deleted subregions in the proximal, middle and distal Yq11, and defined as “AZFa”, “AZFb”, “AZFc” and “AZFd” respectively, with over-lapping between AZFb and AZFc. Each subregion is mainly assessed for microdeletions with corresponding specific sequence-tagged sites (STS) using polymerase chain reaction (PCR) testing. One other technique that may be used is ligase chain reaction (LCR).STSs are single-copy DNA sequences of known mapped location on their respective subregion that serve as markers for genetic and physical mapping of genes. PCR is a technique used to amplify and make copies of a specific DNA region in vitro. In the case of AZF

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locus microdeletions, the absence of one or more STSs, in the same subregion and/or in consecutive ones, detected through PCR, is considered a microdeletion. Most common STSs assessed are:

● AZFa – sY82, sY84, sY86.

● AZFb – sY124, sY127, sY133, sY134, sY143.

● AZFc – sY239, sY242, sY254, sY255, sY1161, sY1201, sY1291, sY1191, sY1206. ● AZFd – sY152, sY145.4,5,6

The AZFc subregion contains repeats of considerable unit lengths, called amplicons, where these amplionic regions are nominated as to their status in microdeletions, according to the specific region and/or the cause of these deletions. These deletions are believed to be responsible for 60% of all Yq deletions. Most common nominations are:

● _{gr/gr – sY1191 is deleted but all flanking markers are present}

● _{b2/b3 –sY1291 is deleted, caused by a gr/gr inversion followed by a b2/b3 deletion or}

by a b2/b3 inversion followed by a gr/gr deletion

● _{b1/b3 – sY1191, sY1197, sY1161 and sY1291 are deleted} ● _{b3/b4 – sY1206 is deleted.}6

Indications for AZF microdeletions screening are based on sperm parameters determined by ejaculate sperm analysis. Alternatively, abnormal sperm parameters are considered to be the only presentation in AZF-related male infertility when no other cause and/or symptom is/are present –

idiopathic cases. According to EAU guidelines, the main sperm disorders found with AZF microdeletions are:

● Non-obstructive azoospermia(azoosp.): complete absence of spermatozoa in semen (most common).

● Oligozoospermia(oligozoosp.): inadequate concentration to cause conception in fertile female; <15 million spermatozoa/Mr. Mild/moderate form is 5-15 million spermatozoa/mL. Severe form is < 5 million spermatozoa/mL. (2nd _{most common).}

● Asthenozoospermia: reduced sperm motility; <32% progressive motile spermatozoa. ● Teratozoospermia: presence of morphologically abnormal sperm; <4% normal forms. ● Oligoasthenoteratozoospermia (oligoasthenoteratozoo.): when oligozoospermia, asthenozoospermia and teratozoospermia are all present.7

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The significance of the AZF locus on the topic of male infertility has been extensively evaluated and analyzed, and a general diagnostic algorithm has already been established. However, when it pertains to idiopathic male infertility and AZF locus microdeletions, the large majority of cases remains unresolved and lacks a clear diagnosis, with the prime suspect at hand. The purposes of this systemic review were to appraise literature discussing idiopathic male infertility, presenting only with abnormal sperm parameters, assessed for AZF microdeletions according to the selection criteria, as case and control populations, deduce the risk relationship between those populations and provide observations and conclusions about the sum of data.

Aim of the thesis: deduce a risk relationship between AZF locus microdeletions, sperm disorders and

male infertility as case and control populations in selected literature.

Objectives of the thesis:

1. Analyze selected studies in terms of assessed AZF subregion and geographical location

2. Analyze the differences between different AZF microdeletion incidences in geographical subregions

3. Determine the risk difference of AZF microdeletion between case and control groups

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METHODOLOGY

This systemic review was performed on the literature of male infertility in AZF regions deletions of the Y-chromosome microdeletions. The literature review was conducted based on the PRISMA-P (preferred reporting items for systemic reviews and meta-analyses protocols) guidelines.8

Search for literature was done through the PUBMED database, with the employment of the Advanced Search Builder (ASB) options. Search terminology was divided across 5 main groups:

● male infertility as a clinical syndrome

● Y chromosome as the primary site of genetic assessment

● AZF locus factor as the specific segment of the Y chromosome being assessed ● Microdeletions as the main disorder of the AZF locus

● Primary sperm disorders as listed according to ICD10 classification The following terminology was deduced, combined and used as follows:

1. “male infertility” OR male infertility OR “human male infertility” OR human male infertility

2. “y chromosome” OR “y-chromosome” OR y chromosome OR “human y chromosome” OR human y chromosome OR “y chromosome long arm” OR “y-chromosome long arm” OR y

chromosome long arm OR y-chromosome long arm OR yq OR yq11.21 OR yq11.22 OR yq11.23 3. “azoospermia factor” OR “azoospermia factor*” OR azf OR azfa OR azfb OR azfc 4. deletion* OR microdeletion* OR “sequence deletion*” OR “gene deletion*”

5. azoospermia* OR aspermia* OR oligozoospermia* OR oligospermia* OR teratozoospermia* OR teratospermia* OR asthenoteratozoospermia* OR astheno

teratozoospermia* OR astheno-teratozoospermia OR oligoasthenoteratozoospermia OR oligo astheno teratozoospermia* OR oligoastheno teratozoospermia OR oligoastheno-teratozoospermia OR oligo-astheno-teratozoospermia* OR globozoospermia* OR “abnormal spermatozoa*” OR abnormal spermatozoa*

After retrieval of search results, results were checked for duplicates. Remaining articles were then screened according to the criteria listed below.

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It was noted that searches were attempted with the same and a simplified version of the search

terminology through several other databases such as Oxford Academic Journals (inadequate results), British Medical Journals (insufficient search parameters) and Wiley Online Library (inadequate results), but searches were omitted with reasons.

Inclusion criteria

The inclusion criteria were as follows: (1) case-control design studies, (2) studies on human males, (3) sufficient sample size, (4) studies analyzing Y chromosome microdeletions of the AZF regions in relation to spermatogenic parameters and subsequent male infertility, (5) gene testing done through STS-PCR analysis, (6) studies within a 10-year range publishing date from 01/04/2019, (7) full-text format

accessible through the Lithuanian University of Health Sciences’ subscribed databases’ function, (8) other studies that begin as case-control studies with information relevant to our inclusion criteria.

Exclusion criteria

The exclusion criteria were as follows: (1) other study designs, (2) studies on animals, (3) studies with a female population as a case population, (4) studies with a publication date older than 10 years, (5) studies including other sex chromosome abnormalities that are not explicitly stated to be excluded or separated – Klinefelter syndrome, Kallman syndrome, abnormal karyotypes, gene polymorphisms and others, (6) studies that assess the relation between Y chromosome deletions and other aspects of diagnosis besides sperm parameters or conditions and diseases other than male infertility, (7) inaccessibility of full texts, (8) and the availability of the full-text articles in the English language.

Data extraction

Detailed data was extracted and summarized independently: primary author’s last name; publication year; country of publication; country of origin (population); total number of men affected with male infertility group (case); total number of non-affected with male infertility men group (control); spermatogenic parameter assessed in population.

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Statistical analysis

Statistical investigation was done using Meta Essentials tool, a set of workbooks for meta-analysis designed for Microsoft Excel.10 _{Analysis of the relationship between AZF locus microdeletions and male}

infertility in idiopathic cases presenting only in abnormal sperm parameters was done according to the Differences between independent group – binary data workbook, with random effects model dichotomous data meta-analytic results; weighting method was inverse variance; model effect size measurement was done in odds ratio (OR) with 95% confidence interval (CI); presentation effect size measurement was done in risk ratio (RR) with 95% CI; statistical significance was done in both one-tailed p-value (p1) and

two-tailed p-value (p2). Research design considered the AZF locus microdeletion factor as the categorical

dependent variable, with the 2 independent factor variables being the infertile case population group and the fertile control population group.

Analysis was begun with mathematical calculation using OR. Produced OR results were then converted into RR results using the tool. The reason behind this process was to provide better conclusions regarding the overall data. OR data was considered mathematically accurate, but ambiguous for conclusion

deduction, whereas RR data was simpler and easier to understand, but mathematically inaccurate in dichotomous data: This has was performed using a proposed formula that uses the mathematically accuracy of OR and the straightforward interpretation of RR. This conversion was available using the tool. Detailed methods, calculations and explanations are available in the Meta Essentials tool.11

Heterogeneity

Cochrane Q-statistics with PQ and I2 values were conducted to assess heterogeneity among studies.

Publication bias

Bias was assessed separately by L’Abbé plot according to random effects model, with inverse variance and log OR.

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RESULTS

Study characteristics

Study characteristic were listed below PRISMA flow chart with reasons for exclusion.

Meta-analysis

13 case-control studies with 5120 case subjects and 2025 controls were used for the meta-analysis of AZF locus microdeletions in idiopathic infertility population with abnormal sperm parameters only.

Analysis showed that, the risk of incidence of any microdeletion in case group is statistically higher compared to the incidence in control group, with an approximate 2-fold (RR = 1.85, CI 95%, p1 = 0.031,

p2 = 0.062). Accordingly, the risk of having a microdeletion between the 2 groups on average is between

0.9 and 3.68 (RR = 1.86, CI 95%, p1 = 0.031, p2 = 0.062).

Heterogeneity was present between the selected studies (CI 95%, Q = 64.2, pQ = 0.000, I2 = 81.31%).

The range of dispersion of the observed effected sizes was between 0.37 and 9.73 (CI 95%, Tau OR= 0.79). 9 studies fell on the side of the statistically significant positive effect, whereas 4 studies fell on the side of the statistically significant negative effect.

Publication bias testing showed that observed effect sizes were asymmetrically distributed around the combined effect size, in the direction of the case group (Figure 2).

88% of all case subjects suffered exclusively from AZFc microdeletions. The rest were divided across the remaining regions.

Distribution of AZFa microdeletions in case population: azoospermia – 76.92%; oligozoospermia –

23.08%. Distribution of AZFb microdeletions in case population: azoospermia – 86.67%; oligozoospermia - 13.33% Distribution of AZFc microdeletions in case population: azoospermia – 50.74%;

oligozoospermia – 33.52%; azoospermia/oligozoospermia – 10.92%; oligoasthenoteratozoospermia – 4.44%. Distribution of AZFd microdeletions in case populations: azoospermia – 50%; oligozoospermia – 50% Distribution of combined microdeletions: azoospermia – 76.09%; oligozoospermia – 17.39%. Distribution of control population microdeletions – 100% AZFc microdeletions. Detailed distribution in control population was available in Table 11.

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Despite all the studies’ approaches to STS-PCR diagnosis being identical, definitions of what was

considered as a microdeletion differed significantly between them, with some considering an STS deletion as a partial deletion (4/13), some as a microdeletion (4/10), and some as a combination (5/10). Studies did not match in terms of assessed AZF subregion per study, with AZFc being the only individually

researched locus between studies (7/13), followed AZFa/b/c (3/13) and AZFa/b/c/d (3/13). Most studies chose azoospermia and oligozoospermia as the assessed sperm parameters (10/13), followed by just azoospermia (2/13) and azoospermia and oligoasthenoteratozoospermia being last (1/13). Inclusion and exclusion criteria of subjects also varied between studies, between subjects enrolled and assessed only by conductors (7/10), subjects previously diagnosed and referred from primary health care centers but not reassessed by conductors (4/13), and subjects previously diagnosed and referred from primary health care centers but reassessed by conductors (2/13). All studies appeared to have a consensus on excluding any possible causes of infertility either from health care centers or study conductors. AZF microdeletions were divided across geographical subregions: Eastern Asia (60.23% of total) – AZFa 2.69%; AZFb 2.42%, AZFc 84.37%, AZFd 0.54%, combined 9.97%; Western Asia (12.99% of total) – AZFb 8%, AZFc 92%; Southern Asia (21.75% of total) – AZFc 100%; South-Eastern Asia (0.81% of total) – AZFa 20%, AZFd 40%, combined 40%; Eastern Europe (1.3% of total) – AZFa 25%, AZFc 50%, combined 25%; Western Europe (2.92% of total) – AZFc 100%.

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Figure 1. PRISMA flow diagram of systemic review

ASB – Advanced Search builder options that were applicable on PudMed database were used after finalized search was performed.120 articles were excluded after abstract review due to difference in topic matter and deemed irrelevant to this review. 4 articles were excluded due to inaccessibility to full-text through the University’s subscribed databases’ function. After full-text review: 3 studies were excluded due to lack of control groups; 3 studies were excluded due to no chromosomal abnormalities testing/exclusion; 2 studies were excluded due to

Id en tif icat ion S cr ee n in g E li gib il ity In clu d ed

PubMed database search (ntotal = 591) → (nASB = 156)

hdfsafsa

Records after duplicate removal (n = 156)

Full-text articles assessed for eligibility (n = 26)

Records screened (n = 156)

Articles included in thesis synthesis (n = 13)

Articles included in meta-analyses synthesis (n = 13)

Records excluded (n = 120)

Full-text articles excluded with reasons (n = 13)

s

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different assessment criteria of haplogroups; 1 study was excluded due to unspecified methodology of subject selection process; 1 study was excluded due to unspecified sperm parameters.26 articles were selected for full-text review. 4 articles were excluded due to inaccessibility to full-text through the University’s subscribed databases’ function. 9 other articles were excluded with reason (3 with no control groups; 3 with no chromosomal

abnormalities testing/exclusion; 2 with different assessment criteria; 1 with unspecified methodology; one with unspecified sperm parameters). Eventually, a total of 13 articles were included

Table 1. Populations and incidences of deletions

Population Deletion No deletion Total Cumulative incidence

Case population 616 4504 5120 616/4548 → %

Control population 237 1871 2025 237/1822 → %

Table 2. Presence and absence of deletions in case and control groups per study

Primary author Case group with deletion Case group without deletion Control group with deletion Control group without deletion L. Alimardanian 2016 10 144 2 109 R. Behulova2011 8 218 0 50 C.G. Beyaz2017 58 275 25 62 J. Choi2012 59 318 14 203 A.A. Hussein 2015 5 51 0 63 X.G. Liu2016 33 133 0 50 R. Mirfakhraie 2010 12 88 0 100 C. Ravel2009 18 346 14 179 S. Sen2015 56 459 16 296 M. Shahid2011 52 366 7 233 H.S. Sin2010 129 266 131 246 V.V. Vijesh2015 26 182 28 97 X.B. Zhu2016 150 1658 0 100

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Table 3. General information of selected studies

Primary author Year Country of

publication

Country of

origin Case population

Control population

L. Alimardanian 2016 United States Iran 154 111

R. Behulova 2011 Czech Republic Slovakia 226 50

C.G. Beyaz 2017 Greece Turkey 333 87

J. Choi 2012 United States South Korea 377 217

A.A. Hussein 2015 United States Malaysia 54 63

X.G. Liu 2016 Brasil China 166 50

R. Mirfakhraie 2010 India Iran 100 100

C. Ravel 2009 United States France 364 193

S. Sen 2015 The Netherlands India 515 312

M. Shahid 2011 United Kingdom India 418 240

H.S. Sin 2010 United Kingdom Japan 395 377

V.V. Vijesh 2015 United States India 208 125

X.B. Zhu 2016 United States China 1808 100

Table 4. Detailed information of selected data

Primary author Spermatogenic parameter AZF subregion Deletion denomination Inclusion/ exclusion Geographical subregion L. Alimardanian

2016 azoosp., oligozoosp. c partial

referred,

assessed Western Asia R. Behulova

2011 azoosp. a, b, c micro assessed

Eastern Europe C.G. Beyaz

2017

azoosp.,

oligoasthenoteratosp. c partial/sub assessed Western Asia J. Choi

2012 azoosp., oligozoosp. c partial assessed Eastern Asia A.A. Hussein

2015 azoosp., oligozoosp. a, b, c, d micro/normal referred

South-Eastern Asia X.G. Liu

2016 azoosp., oligozoosp. a, b, c, d micro referred Eastern Asia R. Mirfakhraie

2010 azoosp. a, b, c, d micro referred Western Asia C. Ravel

2009 azoosp., oligozoosp. c micro/partial assessed

Western Europe S. Sen

2015 azoosp., oligozoosp. c micro/sub

referred,

assessed Southern Asia M. Shahid

2011 azoosp., oligozoosp. c micro/sub assessed Southern Asia H.S. Sin

2010 azoosp., oligozoosp. a, b, c Partial assessed Eastern Asia V.V. Vijesh

2015 azoosp., oligozoosp. c Partial assessed Southern Asia X.B. Zhu

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Table represents spermatogenic parameters assessed, AZF subregions assessed, denomination of deletion of choice by authors, selected inclusion and exclusion criteria of subjects, and the corresponding geographical location of each study.

Table 5. group weight per study; Diagram 1. L’Abbe plot representing table values Case group risk was plotted on x-axis; control group risk was plotted on y-axis. Zero effect reference line was presented in red; observed effect (combined RR between groups) was presented with blue dotted line. Study weights were presented as blue dot. The plot presented the distribution of the studies’ weights on a case/control group risk graph. It was a visual representation of studies’ placement according to their respective bias towards a group against the other. The studies that were on the left side of the observed effect line were considered to favor a stronger case group risk, whereas studies that were on the right side of the observed effect line were considered to favor a stronger control group risk.

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.00 0.10 0.20 0.30 0.40 C ase group ri sk

Control group risk

L'Abbe plot Primary author Control group Case group L. Alimardanian 2016 0.02 0.06 R. Behulova 2011 0.00 0.04 C.G. Beyaz 2017 0.25 0.14 J. Choi 2012 0.06 0.18 A.A. Hussein 2015 0.00 0.09 X.G. Liu 2016 0.00 0.17 R. Mirfakhraie 2010 0.00 0.12 C. Ravel 2009 0.07 0.05 S. Sen 2015 0.05 0.11 M. Shahid 2011 0.03 0.12 H.S. Sin 2010 0.35 0.33 V.V. Vijesh 2015 0.06 0.13 X.B. Zhu 2016 0.00 0.08 Zero effect x y 0.35 0.35 Observed effect x y 0.35 0.4

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Diagram 2. Pictorial representation of forest plot

RR represented the risk in terms on case/control groups, where a negative value would favor a stronger control case risk, and a positive value would favor a stronger case group risk. The CI values represented the ranges of risk in each study from which RR was deduced. The plot represented the ranges as lines with intervals, with weights as blue dots placed on corresponding RR, with an overall representation as well. It was noted that this data was deduced using Log OR, distinctly from the rest of the meta-analytic data.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0.13 0.50 2.00 8.00 32.00 128.00 512.00 Effect Size L. Alimardanian 2016 R. Behulova 2011 C.G. Beyaz 2017 S. Sen 2015 M. Shahid 2011 C. Ravel 2009 R. Mirfakhraie 2010 X.G. Liu 2016 A.A. Hussein 2015 H.S. Sin 2010 V.V. Vijesh 2015 X.B. Zhu 2016 J. Choi 2012 Overall (I2_{=78.3%, p=0)}

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Table 6. Distribution of microdeletions across different AZF subregions in case group Primary author AZFa AZFb AZFc AZFd Combination

L. Alimardanian 2016 10 R. Behulova 2011 2 4 2 C.G. Beyaz 2017 58 J. Choi 2012 59 A.A. Hussein 2015 1 2 2 X.G. Liu 2016 3 3 15 2 10 R. Mirfakhraie 2010 6 1 5 C. Ravel 2009 18 S. Sen 2015 56 M. Shahid 2011 52 H.S. Sin 2010 129 V.V. Vijesh 2015 26 X.B. Zhu 2016 7 6 110 27

Table 7. Distribution of microdeletions across different subregions in control group Primary author AZFa AZFb AZFc AZFd Combination

L. Alimardanian 2016 2 R. Behulova 2011 C.G. Beyaz 2017 25 J. Choi 2012 14 A.A. Hussein 2015 X.G. Liu 2016 R. Mirfakhraie 2010 C. Ravel 2009 14 S. Sen 2015 16 M. Shahid 2011 7 H.S. Sin 2010 131 V.V. Vijesh 2015 28 X.B. Zhu 2016

Combination microdeletions were considered to be a sum of 2 or more deletions from 2 or more distinct AZF subregions.

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Table 8. Distribution of microdeletions across sperm disorders

Sperm parameter AZFa AZFb AZFc AZFd Combinatio n

Azoospermia 10 13 274 2 38

Oligozoospermia 3 2 181 2 8

Azoosp., oligozoosp.* 59

Oligoasthenoterato.* 24

Case group total 13 15 540 4 46

Control group 237

Study of J. Choi 2012 did not distinguish between azoospermia and oligozoospermia subjects.*Study of Beyaz 2017 solely contained oligoasthenoteratozoospermia as a sperm disorder.

Table 9. Distribution of microdeletions across geographical subregions in case group Geographical

subregion

AZFa AZFb AZFc AZFd Combinatio n Eastern Asia 10 9 313 2 37 Western Asia 6 69 5 Southern Asia 134 South-Eastern Asia 1 2 2 Eastern Europe 2 4 2 Western Europe 18

Distribution of each AZF microdeletion with respect to geographical subregions in case population.

Table 10. Distribution of microdeletions across geographical subregions in control group Geographical

subregion

AZFa AZFb AZFc AZFd Combinatio n Eastern Asia 145 Western Asia 27 Southern Asia 51 South-Eastern Asia Eastern Europe Western Europe 14

Distribution of each AZF microdeletion with respect to geographical subregions in control population. All microdeletions were AZFc.

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DISCUSSION

It was determined that was a statistically significant relationship between AZF microdeletions and the incidence of idiopathic male infertility with abnormal sperm parameters only in control population. This relationship was also shown to be statistically stronger than that between AZF microdeletions and fertile male population, with a 1.86-1 ratio. However, the case population had more than double the sample size of the control population. The risk of having a microdeletion within the true population based on the studied population fell in an average between 0.29 and 9.33. In other words, within the real

population, it was estimated that infertile subjects would have a higher risk average between 0.9 and 3.64 than fertile subjects of being affected with a microdeletion. The degree of heterogeneity showed that studies are not from the same population, with 9/13 of all studies showing statistically significant risk of a microdeletion in case population compared to that in control population. Based on one-tailed p-test, it was determined that there was a statistically significant relationship between infertile male population group and the incidence of AZF microdeletion. However, based on two-tailed p-test, it was shown that there a statistically significant relationship between both infertile and fertile male groups on one hand and the incidence of AZF microdeletions on the other.

There has been a significant connection between the incidence of male infertility in idiopathic cases and the presence of AZF microdeletions. In this systemic review, it was found that there is also a significant incidence of AZF microdeletions in fertile males, which has been attributed to the lack of equal control group subjects in all assessed studies, with an estimated range that would suggest a larger affected true population than what was accounted for. In addition, the general consensus of what a microdeletion is did not seem to match across different continents and the investigative algorithm was affected by the selection of what was excluded and included, through which could affect the inclusion of affected case group subjects into appropriate testing procedures. Furthermore, it was found that there was a focus on testing for AZFc microdeletions more than any other locus, which also would not provide an accurate diagnostic basis for all AZF deletions if only one locus is excluded. The inclusion and exclusion criteria of subjects was deemed sufficient, because it was assumed that primary health centers have sufficient

resources to eliminate all other suspicions before referring case subjects to their corresponding studies, similarly to study conductors who performed sufficient tests and procedures. The majority of these studies originated from the Asian continent, while being published in Western journals.

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The highest incidence of AZF microdeletions among case group was Eastern Asia, followed by Southern Asia, Western Asia, Western Europe and Eastern Europe. Knowing that the majority of studies were done in Eastern Asia, the most common microdeletion that was considered sufficient was AZFc across Western, Eastern and Southern Asia (313, 134 and 69 incidences respectively). The highest

incidence of AZFc microdeletions was noted to be in Azoospermia subjects, followed by the control group total. The next closest AZF microdeletion in size was the combination category also being present in the azoospermia category.

27

CONCLUSION

1. Most studies stemmed from Asia, specifically Eastern Asia, while majority of studies assessed AZFc microdeletions exclusively.

2. Across all geographical subregions, AZFc microdeletions were the most prevalent finding 3. The determined risk of suffering male infertility in case group versus control group when

suffering an AZF microdeletion (a, b, c, d and others) was approximately 2:1. 4. AZFc microdeletions accounted for 88% of all case subjects with azoospermia,

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