6de327ba76acdba85e4a0d9ada5b3bc1 pmc_11520404.pdf 1f4edf9213422761c6d9d593a771dd0a28eabd4b pmc_11520404.pdf d8360d2985c94c8eda1927297955f036d8d13dcf865e4ec0a730496ea66d1301 pmc_11520404.pdf Title: Sperm and leukocyte telomere length are related to sperm quality parameters in healthy men from the Led-Fertyl study Subject: Doi: 10.1093/hropen/hoae062 Human Reproduction Open, 2024, 4, 2024 Publication Date: 14/10/2024 Abstract STUDY QUESTION Could sperm and leukocyte telomere length (TL) be associated with sperm quality parameters and reproductive health in men from the general population?SUMMARY ANSWER A positive association between sperm and leukocyte TL with sperm concentration and total count has been demonstrated.WHAT IS KNOWN ALREADY Male factors account for almost half of cases of couple infertility, and shorter TLs have been observed in sperm from men with impaired sperm parameters. However, evidence in men from the general population is limited.STUDY DESIGN, SIZE, DURATION A total of 200 volunteers of reproductive age were recruited between February 2021 and April 2023 to participate in the Lifestyle and Environmental Determinants of Seminogram and Other Male Fertility-Related Parameters (Led-Fertyl) cross-sectional study.PARTICIPANTS/MATERIALS, SETTING, METHODS TLs in sperm and leukocytes were measured using quantitative polymerase chain reaction (qPCR) in 168 and 194 participants, respectively. Sperm parameters, including concentration, total count, motility, vitality, and morphology, were analyzed using a computer-assisted sperm analysis (CASA) SCA® system according to the World Health Organization (WHO) 2010 guidelines. Multivariable regression models were performed to assess the associations between sperm and leukocyte TL, either in tertiles or as continuous variables, and sperm quality parameters while adjusting for potential confounders.MAIN RESULTS AND THE ROLE OF CHANCE Participants in tertiles 2 (T2) and 3 (T3) of sperm TL showed a higher sperm concentration (β: 1.09; 95% CI: 0.09–2.09 and β: 2.06; 95% CI: 1.04–3.09 for T2 and T3, respectively; P-trend < 0.001), compared to those in the reference tertile (T1). Participants in the highest tertile of sperm TL showed higher total sperm count (β: 3.83; 95% CI: 2.08–5.58 for T3 vs T1; P-trend < 0.001). Participants in the top tertile of leukocyte TL showed higher sperm concentration (β: 1.49; 95% CI: 0.44–2.54 for T3 vs T1; P-trend = 0.004), and total count (β: 3.49; 95% CI: 1.62–5.35 for T3 vs T1; P-trend < 0.001) compared with participants in T1. These results remained consistent when sperm and leukocyte TL were modelled as continuous variables.LIMITATIONS, REASONS FOR CAUTION One limitation is the impossibility of establishing a cause–effect relationship due to the cross-sectional study design. Additionally, the sample size of the study cannot be considered large.WIDER IMPLICATIONS OF THE FINDINGS Sperm and leukocyte TLs are associated with sperm quality parameters in the general population. Additional determinations and further studies with larger sample sizes are needed to clarify the mechanisms underlying these associations and to investigate the further implications.STUDY FUNDING/COMPETING INTEREST(S) The Led-Fertyl study was supported by the Spanish government’s official funding agency for biomedical research, Instituto de Salud Carlos III (ISCIII), through the Fondo de Investigación para la Salud (FIS) and co-funded by the European Union ERDF/ESF, ‘A way to make Europe’/‘Investing in your future’ (PI21/01447), and the Diputació de Tarragona (2021/11-No.Exp. 8004330008-2021-0022642). J.S.-S., senior author of the present study, is partially supported by ICREA under the ICREA Academia program. M.F.d.l.P. was supported by a predoctoral grant from the Rovira i Virgili University and Diputació de Tarragona (2020-PMF-PIPF-8). C.V.-H. received a predoctoral grant from the Generalitat de Catalunya (2022 FI_B100108). M.Á.M. was supported by the Sara Borrell postdoctoral fellowship (CD21/00045—Instituto de Salud Carlos III (ISCIII)). All authors declare that they have no conflicts of interest.TRIAL REGISTRATION NUMBER N/A. Keywords: telomere length; sperm quality; male infertility; spermatozoa; leukocyte; reproductive age; Led-Fertyl study Creator: Servigistics Arbortext Advanced Print Publisher 11.1.4667/W Producer: PDFlib+PDI 9.0.7p3 (C++/Win32) CreationDate: Sat Oct 26 08:36:00 2024 CEST ModDate: Sat Oct 26 08:36:07 2024 CEST Custom Metadata: yes Metadata Stream: yes Tagged: yes UserProperties: no Suspects: no Form: none JavaScript: no Pages: 11 Encrypted: no Page size: 612 x 792 pts (letter) Page rot: 0 File size: 1156837 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- KUQWBP+CaeciliaLTStd-Bold Type 1C Custom yes yes yes 1352 0 DFCVDS+CaeciliaLTStd-Roman Type 1C Custom yes yes yes 1357 0 TeX_CM_Roman Type 1 Builtin yes no yes 1362 0 TeX_CM_Maths_Symbols Type 1 Builtin yes no yes 1366 0 JXJIWN+CaeciliaLTStd-Light Type 1C Custom yes yes yes 1370 0 JIYRWJ+CaeciliaLTStd-Heavy Type 1C Custom yes yes yes 1375 0 NILGEH+UniversalStd-NewswithCommPi Type 1C Custom yes yes yes 1380 0 GHPFFT+STIXTwoMath-Regular CID TrueType Identity-H yes yes yes 1385 0 VBEFHU+CaeciliaLTStd-Italic Type 1C Custom yes yes yes 1390 0 TeX_CM_Maths_Italic Type 1 Builtin yes no yes 1395 0 PLOTRP+STIX-Regular Type 1C Custom yes yes yes 1399 0 ZYWOFM+CaeciliaLTStd-LightItalic Type 1C Custom yes yes yes 52 0 DPBFML+TimesNewRomanPSMT Type 1 Custom yes yes no 12 0 EGSDTQ+ArialMT Type 1 Custom yes yes no 16 0 RFAKRB+CaeciliaLTStd-BoldItalic Type 1C Custom yes yes yes 85 0 PSCFFY+STIX-Bold Type 1C Custom yes yes yes 112 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-02-06 02:46:45 CET RepresentationInformation: pmc_11520404.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-12-18 02:31:37 CET Size: 1156837 Format: PDF Version: 1.5 Status: Well-Formed, but not valid SignatureMatches: PDF-hul ErrorMessage: Invalid indirect destination - referenced object 'lhoae062-FM1' cannot be found ID: PDF-HUL-149 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'lhoae062-FM1' cannot be found ID: PDF-HUL-122 Offset: 1012472 MIMEtype: application/pdf Profile: Tagged PDF PDFMetadata: Objects: 1469 FreeObjects: 1 IncrementalUpdates: 2 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: false CenterWindow: false DisplayDocTitle: true NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseOutlines Language: en Outlines: Item: Title: Active Content List Destination: mkchap_artid Children: Item: Title: Introduction Destination: mkchap11__sec Item: Title: Materials and methods Destination: mkchap12__sec Item: Title: Results Destination: mkchap22__sec Item: Title: Discussion Destination: mkchap23__sec Item: Title: Conclusion Destination: mkchap25__sec Item: Title: Supplementary data Destination: mkchap26__sec Item: Title: Data availability Destination: mkchap27__sec Item: Title: Acknowledgements Destination: mkchap28_ack1_ack Item: Title: Authors' roles Destination: mkchap28__sec Item: Title: Funding Destination: mkchap29__sec Item: Title: Conflict of interest Destination: mkchap30__sec Item: Title: References Destination: mkchap31_ref1_ref-list Info: Title: Sperm and leukocyte telomere length are related to sperm quality parameters in healthy men from the Led-Fertyl study Subject: Doi: 10.1093/hropen/hoae062 Human Reproduction Open, 2024, 4, 2024 Publication Date: 14/10/2024 Abstract STUDY QUESTION Could sperm and leukocyte telomere length (TL) be associated with sperm quality parameters and reproductive health in men from the general population?SUMMARY ANSWER A positive association between sperm and leukocyte TL with sperm concentration and total count has been demonstrated.WHAT IS KNOWN ALREADY Male factors account for almost half of cases of couple infertility, and shorter TLs have been observed in sperm from men with impaired sperm parameters. However, evidence in men from the general population is limited.STUDY DESIGN, SIZE, DURATION A total of 200 volunteers of reproductive age were recruited between February 2021 and April 2023 to participate in the Lifestyle and Environmental Determinants of Seminogram and Other Male Fertility-Related Parameters (Led-Fertyl) cross-sectional study.PARTICIPANTS/MATERIALS, SETTING, METHODS TLs in sperm and leukocytes were measured using quantitative polymerase chain reaction (qPCR) in 168 and 194 participants, respectively. Sperm parameters, including concentration, total count, motility, vitality, and morphology, were analyzed using a computer-assisted sperm analysis (CASA) SCA® system according to the World Health Organization (WHO) 2010 guidelines. Multivariable regression models were performed to assess the associations between sperm and leukocyte TL, either in tertiles or as continuous variables, and sperm quality parameters while adjusting for potential confounders.MAIN RESULTS AND THE ROLE OF CHANCE Participants in tertiles 2 (T2) and 3 (T3) of sperm TL showed a higher sperm concentration (β: 1.09; 95% CI: 0.09–2.09 and β: 2.06; 95% CI: 1.04–3.09 for T2 and T3, respectively; P-trend < 0.001), compared to those in the reference tertile (T1). Participants in the highest tertile of sperm TL showed higher total sperm count (β: 3.83; 95% CI: 2.08–5.58 for T3 vs T1; P-trend < 0.001). Participants in the top tertile of leukocyte TL showed higher sperm concentration (β: 1.49; 95% CI: 0.44–2.54 for T3 vs T1; P-trend = 0.004), and total count (β: 3.49; 95% CI: 1.62–5.35 for T3 vs T1; P-trend < 0.001) compared with participants in T1. These results remained consistent when sperm and leukocyte TL were modelled as continuous variables.LIMITATIONS, REASONS FOR CAUTION One limitation is the impossibility of establishing a cause–effect relationship due to the cross-sectional study design. Additionally, the sample size of the study cannot be considered large.WIDER IMPLICATIONS OF THE FINDINGS Sperm and leukocyte TLs are associated with sperm quality parameters in the general population. Additional determinations and further studies with larger sample sizes are needed to clarify the mechanisms underlying these associations and to investigate the further implications.STUDY FUNDING/COMPETING INTEREST(S) The Led-Fertyl study was supported by the Spanish government’s official funding agency for biomedical research, Instituto de Salud Carlos III (ISCIII), through the Fondo de Investigación para la Salud (FIS) and co-funded by the European Union ERDF/ESF, ‘A way to make Europe’/‘Investing in your future’ (PI21/01447), and the Diputació de Tarragona (2021/11-No.Exp. 8004330008-2021-0022642). J.S.-S., senior author of the present study, is partially supported by ICREA under the ICREA Academia program. M.F.d.l.P. was supported by a predoctoral grant from the Rovira i Virgili University and Diputació de Tarragona (2020-PMF-PIPF-8). C.V.-H. received a predoctoral grant from the Generalitat de Catalunya (2022 FI_B100108). M.Á.M. was supported by the Sara Borrell postdoctoral fellowship (CD21/00045—Instituto de Salud Carlos III (ISCIII)). All authors declare that they have no conflicts of interest.TRIAL REGISTRATION NUMBER N/A. Keywords: telomere length; sperm quality; male infertility; spermatozoa; leukocyte; reproductive age; Led-Fertyl study Creator: Servigistics Arbortext Advanced Print Publisher 11.1.4667/W Producer: PDFlib+PDI 9.0.7p3 (C++/Win32) CreationDate: Sat Oct 26 09:06:00 CEST 2024 ModDate: Sat Oct 26 09:06:07 CEST 2024 ID: 0xd4e87c81da664e07e5ac47f4a1f24c51, 0x77fc0d597a05e620cf8b4e2c11ca170c Filters: FilterPipeline: DCTDecode Images: Image: NisoImageMetadata: FormatName: image/jpg CompressionScheme: JPEG ImageWidth: 1890 ImageHeight: 1252 ColorSpace: RGB BitsPerSample: 8 BitsPerSampleUnit: integer Image: NisoImageMetadata: FormatName: image/jpg CompressionScheme: JPEG ImageWidth: 1801 ImageHeight: 1053 ColorSpace: RGB BitsPerSample: 8 BitsPerSampleUnit: integer Fonts: Type0: Font: BaseFont: GHPFFT+STIXTwoMath-Regular Encoding: Identity-H ToUnicode: true Type1: Font: BaseFont: NILGEH+UniversalStd-NewswithCommPi FontSubset: true FirstChar: 1 LastChar: 2 FontDescriptor: FontName: NILGEH+UniversalStd-NewswithCommPi Flags: Nonsymbolic FontBBox: -7, -227, 989, 764 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: KUQWBP+CaeciliaLTStd-Bold FontSubset: true FirstChar: 1 LastChar: 58 FontDescriptor: FontName: KUQWBP+CaeciliaLTStd-Bold Flags: Nonsymbolic, ForceBold FontBBox: -166, -273, 1141, 938 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: DFCVDS+CaeciliaLTStd-Roman FontSubset: true FirstChar: 1 LastChar: 77 FontDescriptor: FontName: DFCVDS+CaeciliaLTStd-Roman Flags: Nonsymbolic FontBBox: -167, -270, 1136, 926 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: VBEFHU+CaeciliaLTStd-Italic FontSubset: true FirstChar: 1 LastChar: 19 FontDescriptor: FontName: VBEFHU+CaeciliaLTStd-Italic Flags: Nonsymbolic, Italic FontBBox: -166, -270, 1053, 915 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: PSCFFY+STIX-Bold FontSubset: true FirstChar: 1 LastChar: 4 FontDescriptor: FontName: PSCFFY+STIX-Bold Flags: Nonsymbolic, ForceBold FontBBox: -998, -552, 2000, 1055 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: TeX_CM_Roman FirstChar: 1 LastChar: 127 FontDescriptor: FontName: TeX_CM_Roman Flags: Symbolic FontBBox: -50, -180, 1000, 720 FontFile: true ToUnicode: true Font: BaseFont: TeX_CM_Maths_Italic FirstChar: 1 LastChar: 127 FontDescriptor: FontName: TeX_CM_Maths_Italic Flags: Symbolic FontBBox: -50, -180, 1000, 720 FontFile: true ToUnicode: true Font: BaseFont: ZYWOFM+CaeciliaLTStd-LightItalic FontSubset: true FirstChar: 1 LastChar: 53 FontDescriptor: FontName: ZYWOFM+CaeciliaLTStd-LightItalic Flags: Nonsymbolic, Italic FontBBox: -166, -267, 1033, 909 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: RFAKRB+CaeciliaLTStd-BoldItalic FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: RFAKRB+CaeciliaLTStd-BoldItalic Flags: Nonsymbolic, Italic, ForceBold FontBBox: -169, -273, 1082, 932 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: TeX_CM_Maths_Symbols FirstChar: 1 LastChar: 138 FontDescriptor: FontName: TeX_CM_Maths_Symbols Flags: Symbolic FontBBox: -50, -180, 1000, 720 FontFile: true ToUnicode: true Font: BaseFont: PLOTRP+STIX-Regular FontSubset: true FirstChar: 1 LastChar: 3 FontDescriptor: FontName: PLOTRP+STIX-Regular Flags: Nonsymbolic FontBBox: -970, -486, 2208, 1023 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: JXJIWN+CaeciliaLTStd-Light FontSubset: true FirstChar: 1 LastChar: 84 FontDescriptor: FontName: JXJIWN+CaeciliaLTStd-Light Flags: Nonsymbolic FontBBox: -164, -266, 1130, 908 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: JIYRWJ+CaeciliaLTStd-Heavy FontSubset: true FirstChar: 1 LastChar: 65 FontDescriptor: FontName: JIYRWJ+CaeciliaLTStd-Heavy Flags: Nonsymbolic, ForceBold FontBBox: -170, -277, 1146, 970 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true CIDFontType2: Font: BaseFont: GHPFFT+STIXTwoMath-Regular CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: GHPFFT+STIXTwoMath-Regular Flags: Nonsymbolic FontBBox: -978, -1641, 3072, 2627 FontFile2: true XMP: uuid:ef488aaf-66f1-2ff9-528f-a7d6dad1c7a5 uuid:CDA2BAC4-C50F-D833-EBE3-FD390F71CAFA 2024-10-26T12:06:00+05:30 2024-10-26T12:06:07+05:30 Servigistics Arbortext Advanced Print Publisher 11.1.4667/W 2024-10-26T12:06:07+05:30 PDFlib+PDI 9.0.7p3 (C++/Win32) telomere length; sperm quality; male infertility; spermatozoa; leukocyte; reproductive age; Led-Fertyl study Sperm and leukocyte telomere length are related to sperm quality parameters in healthy men from the Led-Fertyl study María Fernández de la Puente Cristina Valle-Hita Albert Salas-Huetos María Ángeles Martínez Elena Sánchez-Resino Silvia Canudas Daniel Torres-Oteros Joana Relat Nancy Babio Jordi Salas-Salvadó Doi: 10.1093/hropen/hoae062 Human Reproduction Open, 2024, 4, 2024 Publication Date: 14/10/2024 Abstract STUDY QUESTION Could sperm and leukocyte telomere length (TL) be associated with sperm quality parameters and reproductive health in men from the general population?SUMMARY ANSWER A positive association between sperm and leukocyte TL with sperm concentration and total count has been demonstrated.WHAT IS KNOWN ALREADY Male factors account for almost half of cases of couple infertility, and shorter TLs have been observed in sperm from men with impaired sperm parameters. However, evidence in men from the general population is limited.STUDY DESIGN, SIZE, DURATION A total of 200 volunteers of reproductive age were recruited between February 2021 and April 2023 to participate in the Lifestyle and Environmental Determinants of Seminogram and Other Male Fertility-Related Parameters (Led-Fertyl) cross-sectional study.PARTICIPANTS/MATERIALS, SETTING, METHODS TLs in sperm and leukocytes were measured using quantitative polymerase chain reaction (qPCR) in 168 and 194 participants, respectively. Sperm parameters, including concentration, total count, motility, vitality, and morphology, were analyzed using a computer-assisted sperm analysis (CASA) SCA® system according to the World Health Organization (WHO) 2010 guidelines. Multivariable regression models were performed to assess the associations between sperm and leukocyte TL, either in tertiles or as continuous variables, and sperm quality parameters while adjusting for potential confounders.MAIN RESULTS AND THE ROLE OF CHANCE Participants in tertiles 2 (T2) and 3 (T3) of sperm TL showed a higher sperm concentration (β: 1.09; 95% CI: 0.09–2.09 and β: 2.06; 95% CI: 1.04–3.09 for T2 and T3, respectively; P-trend < 0.001), compared to those in the reference tertile (T1). Participants in the highest tertile of sperm TL showed higher total sperm count (β: 3.83; 95% CI: 2.08–5.58 for T3 vs T1; P-trend < 0.001). Participants in the top tertile of leukocyte TL showed higher sperm concentration (β: 1.49; 95% CI: 0.44–2.54 for T3 vs T1; P-trend = 0.004), and total count (β: 3.49; 95% CI: 1.62–5.35 for T3 vs T1; P-trend < 0.001) compared with participants in T1. These results remained consistent when sperm and leukocyte TL were modelled as continuous variables.LIMITATIONS, REASONS FOR CAUTION One limitation is the impossibility of establishing a cause–effect relationship due to the cross-sectional study design. Additionally, the sample size of the study cannot be considered large.WIDER IMPLICATIONS OF THE FINDINGS Sperm and leukocyte TLs are associated with sperm quality parameters in the general population. Additional determinations and further studies with larger sample sizes are needed to clarify the mechanisms underlying these associations and to investigate the further implications.STUDY FUNDING/COMPETING INTEREST(S) The Led-Fertyl study was supported by the Spanish government’s official funding agency for biomedical research, Instituto de Salud Carlos III (ISCIII), through the Fondo de Investigación para la Salud (FIS) and co-funded by the European Union ERDF/ESF, ‘A way to make Europe’/‘Investing in your future’ (PI21/01447), and the Diputació de Tarragona (2021/11-No.Exp. 8004330008-2021-0022642). J.S.-S., senior author of the present study, is partially supported by ICREA under the ICREA Academia program. M.F.d.l.P. was supported by a predoctoral grant from the Rovira i Virgili University and Diputació de Tarragona (2020-PMF-PIPF-8). C.V.-H. received a predoctoral grant from the Generalitat de Catalunya (2022 FI_B100108). M.Á.M. was supported by the Sara Borrell postdoctoral fellowship (CD21/00045—Instituto de Salud Carlos III (ISCIII)). All authors declare that they have no conflicts of interest.TRIAL REGISTRATION NUMBER N/A. © The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. telomere length sperm quality male infertility spermatozoa leukocyte reproductive age Led-Fertyl study application/pdf PStill version 1.84.42 True 3 A http://ns.adobe.com/pdf/1.3/ pdf Adobe PDF Schema internal A name object indicating whether the document has been modified to include trapping information Trapped Text http://ns.adobe.com/xap/1.0/mm/ xmpMM XMP Media Management Schema internal UUID based identifier for specific incarnation of a document InstanceID URI internal The common identifier for all versions and renditions of a document. OriginalDocumentID URI http://www.aiim.org/pdfa/ns/id/ pdfaid PDF/A ID Schema internal Part of PDF/A standard part Integer internal Amendment of PDF/A standard amd Text internal Conformance level of PDF/A standard conformance Text Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 168, 593, 171, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 173, 593, 176, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 178, 593, 181, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 258, 593, 267, 603 Flags: Print Annotation: Subtype: Link Rect: 267, 593, 271, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 272, 593, 276, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 277, 593, 280, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 362, 593, 372, 603 Flags: Print Annotation: Subtype: Link Rect: 372, 593, 375, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 377, 593, 380, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 381, 593, 385, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 386, 593, 390, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 391, 593, 395, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 489, 593, 493, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 494, 593, 497, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 499, 593, 502, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 504, 593, 507, 601 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 139, 584, 143, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 144, 584, 147, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 209, 583, 219, 593 Flags: Print Annotation: Subtype: Link Rect: 219, 584, 223, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 224, 584, 231, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 232, 584, 236, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 320, 584, 323, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 325, 584, 331, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 378, 584, 381, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 383, 584, 386, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 387, 584, 394, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 447, 583, 457, 593 Flags: Print Annotation: Subtype: Link Rect: 457, 584, 460, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 462, 584, 465, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 467, 584, 470, 592 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 472, 584, 475, 592 Flags: Print Destination: null Annotation: Subtype: Link Rect: 130, 574, 140, 584 Flags: Print Annotation: Subtype: Link Rect: 140, 574, 144, 582 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 145, 574, 149, 582 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 150, 574, 153, 582 Flags: Print Destination: 1335 Annotation: Subtype: Link Rect: 155, 574, 158, 582 Flags: Print Destination: null Annotation: Subtype: Link Rect: 322, 429, 329, 436 Flags: Print Annotation: Subtype: Link Rect: 331, 429, 449, 435 Flags: Print Annotation: Subtype: Link Rect: 214, 411, 221, 418 Flags: Print Annotation: Subtype: Link Rect: 222, 411, 341, 417 Flags: Print Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 53, 374, 131, 382 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 143, 352, 269, 359 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 84, 329, 158, 337 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 129, 285, 223, 293 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 187, 251, 238, 259 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 204, 229, 264, 237 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 285, 196, 294, 204 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 51, 185, 96, 192 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 220, 162, 279, 170 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 152, 118, 214, 126 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 53, 73, 179, 81 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 417, 400, 550, 408 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 314, 286, 382, 294 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 488, 286, 553, 294 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 559, 286, 559, 294 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 312, 275, 389, 282 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 443, 275, 502, 282 Flags: Print Destination: 45 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 148, 301, 279, 308 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 370, 462, 486, 470 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 443, 262, 556, 269 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 478, 85, 556, 93 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 445, 63, 556, 70 Flags: Print Destination: 45 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 447, 662, 555, 670 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 312, 651, 329, 658 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 312, 562, 342, 570 Flags: Print Destination: 9 Annotation: Subtype: Link Rect: 507, 494, 535, 501 Flags: Print Destination: 23 Annotation: Subtype: Link Rect: 481, 459, 513, 467 Flags: Print Destination: 23 Annotation: Subtype: Link Rect: 533, 459, 538, 467 Flags: Print Destination: 27 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 320, 664, 403, 672 Flags: Print Annotation: Subtype: Link Rect: 328, 653, 357, 661 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 379, 653, 400, 661 Flags: Print Destination: 35 Annotation: Subtype: Link Rect: 320, 542, 348, 550 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 408, 520, 429, 528 Flags: Print Destination: 35 Annotation: Subtype: Link Rect: 398, 421, 426, 429 Flags: Print Destination: 40 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 180, 363, 200, 371 Flags: Print Destination: 40 Annotation: Subtype: Link Rect: 172, 107, 294, 115 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 51, 96, 68, 104 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 210, 62, 289, 70 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 426, 374, 494, 382 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 499, 374, 555, 382 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 312, 363, 329, 371 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 405, 296, 480, 304 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 342, 207, 412, 215 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 449, 185, 517, 193 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 338, 62, 412, 70 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 314, 51, 375, 59 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 380, 51, 441, 59 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 415, 40, 474, 48 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 479, 40, 550, 48 Flags: Print Destination: 45 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 161, 305, 223, 313 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 95, 283, 166, 291 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 149, 173, 258, 180 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 203, 117, 295, 125 Flags: Print Destination: 45 Annotation: Subtype: Link Rect: 140, 669, 143, 676 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 169, 660, 172, 667 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 147, 624, 150, 631 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 168, 409, 172, 416 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 63, 344, 72, 350 Flags: Print Destination: 31 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 51, 271, 133, 278 Flags: Print Annotation: Subtype: Link Rect: 188, 124, 266, 131 Flags: Print Annotation: Subtype: Link Rect: 274, 124, 294, 131 Flags: Print Annotation: Subtype: Link Rect: 51, 112, 102, 120 Flags: Print Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 153, 669, 157, 676 Flags: Print Destination: 40 Annotation: Subtype: Link Rect: 169, 660, 172, 667 Flags: Print Destination: 40 Annotation: Subtype: Link Rect: 147, 624, 150, 631 Flags: Print Destination: 40 Annotation: Subtype: Link Rect: 168, 409, 172, 416 Flags: Print Destination: 40 Annotation: Subtype: Link Rect: 63, 344, 72, 350 Flags: Print Destination: 40 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 51, 470, 294, 477 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 62, 459, 292, 466 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 51, 437, 294, 444 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 62, 426, 294, 433 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 62, 415, 294, 422 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 62, 404, 294, 411 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 62, 393, 163, 400 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 51, 382, 292, 390 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 51, 360, 294, 368 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 62, 349, 208, 357 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 51, 338, 294, 346 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 62, 327, 294, 335 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 62, 316, 294, 324 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 62, 305, 90, 313 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 51, 294, 294, 302 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 62, 283, 294, 291 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 62, 272, 128, 280 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 51, 261, 294, 269 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 62, 251, 294, 258 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 62, 240, 294, 247 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 62, 229, 203, 236 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 51, 218, 294, 225 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 62, 207, 294, 214 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 62, 196, 294, 203 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 62, 185, 294, 192 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 62, 174, 142, 181 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 51, 163, 294, 170 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 62, 152, 294, 159 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 62, 141, 294, 148 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 62, 130, 134, 137 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 51, 119, 294, 126 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 62, 108, 294, 116 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 62, 97, 189, 105 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 235, 97, 294, 105 Flags: Print Annotation: Subtype: Link Rect: 62, 86, 162, 94 Flags: Print Annotation: Subtype: Link Rect: 312, 731, 555, 738 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 324, 719, 553, 727 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 312, 697, 555, 704 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 324, 685, 555, 693 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 324, 674, 555, 681 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 324, 663, 473, 670 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 312, 652, 555, 659 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 640, 555, 648 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 629, 409, 636 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 312, 618, 555, 625 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 324, 606, 555, 614 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 324, 595, 555, 602 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 324, 583, 553, 591 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 312, 561, 555, 568 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 324, 550, 555, 557 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 324, 538, 555, 546 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 324, 527, 415, 534 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 312, 516, 555, 523 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 504, 555, 512 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 493, 553, 500 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 312, 470, 555, 478 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 324, 459, 505, 466 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 312, 448, 555, 455 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 437, 555, 444 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 425, 555, 433 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 414, 553, 421 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 312, 391, 555, 399 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 324, 380, 555, 387 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 324, 368, 555, 376 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 324, 357, 377, 364 Flags: Print Destination: 31 Annotation: Subtype: Link Rect: 312, 346, 555, 353 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 334, 553, 342 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 312, 312, 555, 320 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 301, 396, 308 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 312, 290, 555, 297 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 278, 555, 286 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 267, 555, 274 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 255, 411, 263 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 312, 244, 555, 252 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 233, 393, 240 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 414, 233, 555, 240 Flags: Print Annotation: Subtype: Link Rect: 324, 222, 423, 229 Flags: Print Annotation: Subtype: Link Rect: 312, 211, 555, 218 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 324, 199, 555, 207 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 324, 188, 468, 195 Flags: Print Destination: 5 Annotation: Subtype: Link Rect: 312, 177, 555, 184 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 324, 165, 519, 173 Flags: Print Destination: 1 Annotation: Subtype: Link Rect: 312, 154, 555, 162 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 324, 143, 555, 150 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 324, 131, 462, 139 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 312, 120, 555, 128 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 324, 109, 555, 117 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 324, 98, 555, 105 Flags: Print Destination: 27 Annotation: Subtype: Link Rect: 324, 86, 413, 94 Flags: Print Destination: 27 Page: Sequence: 11 Checksum: 6fa4b0ec Type: CRC32 Checksum: 6de327ba76acdba85e4a0d9ada5b3bc1 Type: MD5 Checksum: 1f4edf9213422761c6d9d593a771dd0a28eabd4b Type: SHA-1 Checksum: d8360d2985c94c8eda1927297955f036d8d13dcf865e4ec0a730496ea66d1301 Type: SHA-256