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PESA/MESA/TESA/TESE Sperm Processing

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In Vitro Fertilization

Abstract

In men with azoospermia, spermatozoa can be retrieved from the epididymis or testis using different surgical methods such as PESA, TESA, TESE, and micro-TESE. After collecting the epididymal fluid or testicular tissue, laboratory techniques are used to remove contaminants, cellular debris, harmful microorganisms, and red blood cells. Processed spermatozoa can be utilized immediately for intracytoplasmic sperm injection or cryopreserved for future use. The surgically retrieved gametes are more fragile than the ejaculated ones. As such, sperm processing techniques should be used with care to avoid jeopardizing the sperm fertilizing potential. In this chapter, we describe the methods for optimal processing of surgically retrieved specimens and present our clinical experience with ICSI using testicular and epididymal sperm in azoospermic men. Additionally, we provide essential tips about how to optimally handle surgically retrieved sperm.

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Author information

Authors and Affiliations

  1. ANDROFERT, Andrology & Human Reproduction Clinic, Referral Center for Male Infertility, Campinas, SP, Brazil

    Sidney Verza Jr.

  2. ANDROFERT, Andrology and Human Reproduction Clinic, Campinas, SP, Brazil

    Sandro C. Esteves

Authors
  1. Sidney Verza Jr.
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  2. Sandro C. Esteves
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Corresponding author

Correspondence to Sandro C. Esteves .

Editor information

Editors and Affiliations

  1. Reproductive Biology Associates – Prelude, Atlanta, GA, USA

    Zsolt Peter Nagy

  2. Astra Fertility Group, Mississauga, ON, Canada

    Alex C. Varghese

  3. Cleveland Clinic, Cleveland, OH, USA

    Ashok Agarwal

Appendices

Appendix

  • Erythrocyte Lysing Buffer Solution (ELBS) : 155 mM NH4Cl + 10 mM KHCO3 + 2 mM EDTA dissolved in sterile water. Adjust the pH to 7.2, if necessary. Upon finishing the first dilution and centrifugation step, resuspend the pellet with 2.0 mL of ELBS and keep the mixture at room temperature for 10 min. Then, centrifuge the sample at ×300 g for 5 min, discharge the supernatant, and resuspend the pellet in 0.2 mL of fresh HEPES-buffered protein-supplemented sperm medium [46].

  • Enzymatic Digestion : Prepare 1.0 mL of HEPES-buffered Human Tubal Fluid (Modified HTF culture medium, cat.# 90126, Irvine Scientific, USA) supplemented with 5% of Human Serum Albumin (HSA, cat.# 9988, Irvine Scientific, USA) and 2.6 mg of collagenase type IV (Sigma C5138; activity: 378 Units/mg solid) [64, 65]. Upon finishing the first dilution and centrifugation step, resuspend the pellet with 1.0 mL of the enzymatic digestion solution and incubate at 37 °C for 1 h. Homogenize the suspension every 10–15 min during the incubation period to obtain better enzymatic digestion of the tissue. The digested tissue solution is centrifuged for 5 min at 300 g, and the pellet is resuspended in 100–200 μL of Modified HTF culture medium supplemented with 5% of HSA.

  • Hyposmotic Solution (HOS) : Prepare a 150-mOsm/kg HOS solution by dissolving 7.35-mg sodium citrate and 13.51-mg fructose in 1-mL sterile reagent water [27]. Alternatively, a 139-mOsm/kg HOS solution can be prepared by mixing 1-mL sperm medium to 1-mL sterile reagent water [25, 26].

  • Pentoxifylline Solution (PF) : Prepare a 5-mM solution of PF by dissolving 1.391-mg pentoxifylline (Sigma cat.# P-1784) in 1 mL of HEPES-buffered culture medium [30].

Review Questions

  1. 1.

    What is the difference between PESA, MESA, TESA, TESE, and micro-TESE and when each method is indicated?

  2. 2.

    Which materials, equipment, reagents, and IVF laboratory setup are needed to perform PESA, MESA, TESA, TESE, and micro-TESE?

  3. 3.

    Which additional sperm processing techniques can be used during sperm search to improve the chances of finding sperm in specimens from non-obstructive azoospermic patients?

  4. 4.

    Which are the additional methods for selecting viable sperm for ICSI when only immotile spermatozoa are obtained after PESA/MESA/TESA/TESE/micro-TESE?

  5. 5.

    What are the advantages of using micro-TESE as the method of choice for harvesting sperm in men with non-obstructive azoospermia in preference over TESE or TESA?

  6. 6.

    What is the association between testicular histopathology results and wet tissue examination in retrieved testicular specimens?

  7. 7.

    Is sperm fertilizing potential similar in obstructive and non-obstructive azoospermia?

  8. 8.

    How to optimize sperm cryopreservation in cases in which only a few spermatozoa are recovered?

Glossary

Azoospermia

Absence of spermatozoa in the microscopic examination of the seminal fluid after centrifugation on at least two separate occasions.

Cryopreservation

The freezing process for storage of gametes or gonadal tissue at ultra-low temperature.

ICSI

Intracytoplasmic Sperm Injection: a procedure in which a single spermatozoon is injected into the oocyte cytoplasm.

MESA

Microsurgical Epididymal Sperm Aspiration: a microsurgical procedure used to aspirate spermatozoa directly from the epididymal tubules for use in an ICSI procedure.

Micro-TESE

Microdissection Testicular Sperm Extraction: a microsurgical procedure used to dissect the seminiferous tubules within the testis in an attempt to identify areas of sperm production and extract spermatozoa for use in an ICSI procedure.

PESA

Percutaneous Epididymal Sperm Aspiration: a procedure in which a needle is inserted into the epididymis to retrieve spermatozoa for use in an ICSI procedure.

Sperm Processing

Laboratory techniques used to remove contaminants (cellular debris, microorganisms, red blood cells, etc.) and to select the best-quality spermatozoa to be used in conjunction to assisted reproduction technology.

TESA

Testicular Sperm Aspiration: a procedure in which a needle is inserted into the testis in order to retrieve spermatozoa for use in an ICSI procedure.

TESE

Testicular Sperm Extraction: operative removal of testicular tissue in an attempt to collect sperm for use in an ICSI procedure.

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Verza, S., Esteves, S.C. (2019). PESA/MESA/TESA/TESE Sperm Processing. In: Nagy, Z., Varghese, A., Agarwal, A. (eds) In Vitro Fertilization. Springer, Cham. https://doi.org/10.1007/978-3-319-43011-9_26

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