Indian Journal of Obstetrics and Gynecology Research

Print ISSN: 2394-2746

Online ISSN: 2394-2754

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Indian Journal of Obstetrics and Gynecology Research (IJOGR) open access, peer-reviewed quarterly journal publishing since 2014 and is published under auspices of the Innovative Education and Scientific Research Foundation (IESRF), aim to uplift researchers, scholars, academicians, and professionals in all academic and scientific disciplines. IESRF is dedicated to the transfer of technology and research by publishing scientific more...


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Sinha, Patel, Bagde, Sinha, and Joshi: Observational study of Y chromosome microdeletion, EAA markers and non EAA markers in Chhattishgarh


Introduction

Outcome of pregnancy depends on the characteristics of male and female gametes. If one of them is not in existence or of not good quality, fertilization or quality of embryo formation would be affected. Male factor is responsible for approximately 50% of total infertility. Y chromosome microdeletion is an important factor in the occurrence of male infertility. There are many STS markers available to detect Y chromosome microdeletion. Usually six markers are tested at initial stage after semenogram that are recommended by European Academy of Andrology (EAA) society.1 However other markers may be deleted even if EAA markers are not deleted. Therefore, testing of EAA markers may not serve the purpose in Indian population. Important non EAA markers should also tested along with EAA markers.

The development of male gamete takes place in various stages. Primordial germ cells which form in ectoderm of the bilaminer germ disc of human embryo and move to the wall of yolk sac give rise to spermatogonia. Spermatogonia converts in to primary spermatocytes by repeated mitotic division. Primary spermatocyte by meiotic division(I) develops secondary spermatocytes. Each secondary spermatocytes by meiotic division(II) forms two spermatids. The spermatids becomes spermatozoa by the process of spermiogenesis. This is the final stage in maturation. Overall process of spermatogenesis is regulated by gene present on Y chromosome. Quantity and quality of sperm depends on partial and complete deletion of genes.

Aim of our study is to determine the frequency of the Y chromosome microdeletion in idiopathic infertile men using both EAA and non EAA markers in central region of India. This microdeletion test is important for counseling of couples who opt for assisted reproductive technology (ART) as this defect in gene may get transferred to the offspring.

Materials and Methods

A total of forty men of Chhattisgarh origin presenting to infertility clinic seeking treatment of infertility were recruited in the study. Thirty normal fertile men of the same origin were enrolled as control. All infertile men and fertile men were of same age group ranging from 22-45 years. Infertile men in principle met the definition of infertility ie one year of unprotected intercourse and not leading to conception (regardless of fertility of partners). These were subjected to comprehensive questionnaire regarding their life style habits like smoking, alcohol and drug abuse, medical history, surgical history, marital age, sexual and family history. Every individual made available a minimum of two semen sample with abstinence period of three days. These specimens were evaluated on the basis of criteria of WHO 2010.2 Informed consent was obtained from every couple. On the basis of mean sperm count, infertile men were grouped in azoospermia, severe oligozoospermia, oligzoospermia and normozoospermia. In addition, blood samples were obtained for DNA extraction.

Genomic DNA was obtained from Blood lymphocytes using Qiagen kit. Primers were designed for EAA markers sY86, sY 127, sY254, sY84, sY 134, sY255 and Non- EAA markers sY746, sY182, sY121, sY128, sY130, sY143, sY145, sY160 (Table 1). All PCR reactions were performed in a total reaction volume 13µl. PCR were in simplex or multiplex fashion. Additional STS marker for internal control was SRY gene. Fertile male and female DNAs were used as external controls. Five microliter of genomic DNA, 1X amplification buffer, mM dNTPs, 10-25 pmol of each primer and 1.25 IU of taq polymerase were used in reaction mixture. After initial denaturation 5min, annealing reaction was carried out at a temperature specific for that primer. PCR products were separated in 1.2- 2% agarose by gel electrophoresis.

Result

All cytogenetically normal infertile and fertile patients were subjected for deletion testing. Y chromosome microdeletion were confirmed for eleven STS markers. Eleven subjects showed deletion out of forty cases whereas no deletion was observed in controls. Out of eleven cases, two (18%) showed deletion for more than one loci (Table 2, Table 3). Single deletion of AZF a,b,c were 5/11, 3/11, 1/11 respectively (Figure 1). Double deletions of AZF a+c and b+c were observed in 1/11, 1/11 respectively (Figure 2). Total oligozoospic cases, severe oligozoospermic cases and azoospermic cases were 17/40, 11/40 and 12/40 respectively. Over all frequency of deletion in AZFa, AZFb and AZFc regions were found 7(12.5%), 4(7.5%) and 2(2.5%) cases respectively.

Table 1

Showing Non EAA markers and their primer sequence

Primers

Forward sequence

Reverse sequence

sY746

5’-TTGACTGCTTATTCTACACAA-3’

5’-CAGGGGAAATTGGGTTTT-3’

sY182

5’-TCAGAAGTGAAACCCTGTATG-3’

5’-GCATGTGACTCAAAGTATAAGC-3’

sY121

5’-AGTTCACAGAATGGAGCCTG-3’

5’-CCTGTGACTCCAGTTTGGTC-3’

sY128

5’-GGATGAGACATTTTTGTGGG-3’

5’-AGCCCAATGTAAACTGGACA-3’

sY130

5’-AGAGAGTTTTCTAACAGGGCG-3’

5’-TGGGAATCACTTTTGCAACT-3’

sY143

5’-GCAGGATGAGAAGCAGGTAG -3’

5’-CCGTGTGCTGGAGACTAATC-3’

sY145

5’-CAACACAAAAACACTCATATACTCG-3’

5’-TTGAGAATAATTGTATGTTACGGG-3’

sY160

5’-TACGGGTCTCGAATGGAATA-3’

5’-TCATTGCATTCCTTTCCCATT-3’

Table 2

Different Primer tested and cases showing deletions

S.No.

STS markers

Deletions in case

Bp

Locus

1

sY86

No

320

AZFa

EAA STS markers

2

sY 127

C13

274

AZFb

3

sY254

C13

380

AZFc

4

sY84

No

326

AZFa

5

sY 134

C13

301

AZFb

6

sY255

C13

126

AZFc

7

sY746

C28, C39

216

AZFa

Non EAA markers

8

sY182

C11, C15, C22, C32

125

AZFa

9

sY121

No

190

AZFb

10

sY128

C13

228

AZFb

11

sY130

C13, C20

173

AZFb

12

sY143

C4, C31,

311

AZFb

13

sY145

C22,

143

AZFc

14

sY160

C27,

236

AZFc

16

sY14 SRY

No

476

Table 3

Cases, deleted regions and semen condition

S.No.

Case no

Deleted AZF region

Semenogram

1

C4

AZFb

Oligozoospermia

2

C13

AZFb+c

Azoospermia

3

C11

AZFa

Sever Oligozoospermia

4

C15

AZFa

Sever Oligozoospermia

5

C20

AZFb

Oligozoospermia

6

C22

AZFa+c

Oligozoospermia

7

C27

AZFc

Azoospermia

8

C28

AZFa

Oligozoospermia

9

C31

AZFb

Oligozoospermia

10

C32

AZFa

Oligozoospermia

11

C39

AZFa

Sever Oligozoospermia

Figure 1

Agarose gel analysis showing microdeletions of STS markers

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/eb7d9452-f7bc-40a0-9183-26cfda1bfe32/image/f9498dfc-07b7-4cb6-8739-d0f58df83ea0-uimage.png

Figure 2

Percentage deletion of AZF region in eleven subjects

https://typeset-prod-media-server.s3.amazonaws.com/article_uploads/eb7d9452-f7bc-40a0-9183-26cfda1bfe32/image/139aa5e9-1ad2-406b-9225-57cf0259253a-uimage.png

Discussion

Many causes of infertility are treatable whereas many are not. Most of the genetic causes are not treatable and irreversible. Many males presenting without sperm in ejaculate have deletion of Ychromosome region. These deleted regions include azoospermia factor (AZF) gene. This AZF gene is located in long arm of Y chromosome. Tiepolo and Zuffardi3 first localized factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm.

In earlier studies, there is distinguished variation in microdeletion frequency. These variations are depending on the selection criteria of patient, number of markers used, semenogram, and cytogenetic variability in studies. Arrest in different stages of spermetogenesis is reflection Yq microdeletion. The microdeletion in AZFa region is associated with SOC syndrome and azoospermia. The microdeletion of AZFb region is associated with meiotic maturation arrest. The microdeletion of AZFc region is associated with arrest at spermatid stage or hypospermatogenesis.4, 5

The microdeletion of AZFb region is associated with hypospermatogenesis. Most common deletion observed in our study was AZFa. These individuals were oligozoospermic and severe oligozoospermic. Deletion in AZFa region in oligozoospermic and azoospermic men were common.6, 7 Studies show highest frequency of deletion was AZFc type.8, 9 In our study it was AZFa type which may be due to small sample size.

Double deletion was observed in two subjects; AZFa+c and AZFb+c (Table 3). Deletion type AZFa+c is rare. AZFa+c was observed in oligozoospermic case. Only complete deletion of AZFa is associated with absence of sperm.10

The AZFb microdeletion is associated with developmental arrest of germ cells at spermatid stage and resulted to meiotic maturation arrest. AZFc deletion is associated with arrest of germ cell development at spermatid stage and hypospermatogenesis with sperm count.

Sen et al. stated that frequency of deletion was significantly high in the group using both EAA and non EAA markers.11 Based on this analysis approximately 3.1% cases would be missed if only EAA markers are used. In our study, only one case exhibited deletion of EAA markers out of forty cases and it was double deletion.

Conclusion

These findings add to the growing literature of Yq microdeletion. Both EAA markers and non EAA markers should be tested for Yq microdeletion. Frequency of EAA markers deletion was found in one case out of forty cases and it was double deletion. Very less frequency of Yq microdeletion was observed with EAA markers. Chances of missing of infertility cases can happen. This test is important to screen the patients who are opting assisted reproductive techniques as it may be transmitted in offspring.

Source of Funding

Authors are thankful to Chhattisgarh council of science and technology for funding the project (grant number-Endt. No 7068/ CCOST/MRP/2018).

Conflict of Interest

None.

References

1 

M Simoni E Bakker C Krausz EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions: State of the artInt J Androl2013272409

2 

World Health Organization. WHO laboratory manual for the examination and processing of human semen. 5th ed. United Kingdom2010

3 

L Tiepolo O Zuffardi Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long armHum Genet19763411924

4 

NN Chai EC Salido PH Yen Multiple functional copies of RBM gene family, a spermatogenesis candidate gene family, a spermatogenesis candidate on the human Y chromosomeGenomics19974535561

5 

L Delbridge JL Harry R Toder RJW O’ Neill K Ma AC Chandley A human candidate spermatogenesis gene, RBM1, is conserved and amplified on the marsupial Y chromosomeNat Genet 1997152131610.1038/ng0297-131

6 

C Foresta E Moro A Ferlin Y chromosome microdeletions and alterations of spermatogenesisEndocr Rev20012222263910.1210/edrv.22.2.0425.

7 

C Krausz S Degl’Innocenti Y chromosome and male infertilityFront Biosci 200611304961

8 

S Sen P Ambulkar I Hinduja K Zaveri J Gokral A Pal Susceptibility of gr/gr rearrangements to azoospermia or oligozoospermia is dependent on DAZ and CDY1 gene copy deletionsJ Assist Reprod Genet201532133341

9 

PS Ambulkar SS Pande Study of ychromosome microdeletion in azoospermic Infertile Males using Multiplex PCR AnalysisBiosci Biotech Res Asia201815210.13005/bbra/2639

10 

C Kampa P Hrischmann H Voss K Huellen PH Vogt Two long homologous retroviral sequence blocks in proximal Yq11 causes AZFa microdeletion as a result of intrachromosomal recombinationHum Mol Gent20009256372

11 

S Sen AR Pasi R Dada M Shamsi D Modi Y chromosome microdeletions in infertile men: prevalence, phenotypes and screening markers for the Indian populationJ Assisted Reprod Genet20143041322



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© This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Article type

Original Article


Article page

310-313


Authors Details

Manisha B Sinha*, Suprava Patel, Nilaj Bagde, H P Sinha, Apoorva Joshi


Article History

Received : 24-12-2020

Accepted : 03-05-2021

Available online : 25-08-2021


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