Atlas of Assisted Reproductive Technologies 9819900182, 9789819900183

This atlas provides details of the procedures of assisted reproductive techniques. The science of in-vitro fertilization

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Atlas of Assisted Reproductive Technologies
 9819900182, 9789819900183

Table of contents :
Part I: ART Laboratory and Equipment
Set Up of an IVF Lab
1 Non-Sterile Area
2 Sterile Area
3 Infrastructure of the IVF Laboratory
4 HEPA Filters (High-Efficiency Particulate Air) (Fig. 22)
5 Air Handling Unit (AHU) (Fig. 23)
Equipment for Micromanipulation
1 ICSI Instrumentation
1.1 Main Components
1.2 Types of Micromanipulators: Micromanipulators can be of three types
1.3 Function of Manipulators
1.3.1 Hydraulic Micromanipulator Components and Their Function
1.4 Eppendrof Micromanipulator: Fully Motorized
Part II: Clinical Procedures
Ultrasound Imaging of the Ovary in Infertility
Oocyte Retrieval
1 Equipment and Consumables Required for Ovum Pick Up
2 Patient Preparation and Anaesthesia
3 Preparation of the Ultrasound Machine, the Probe and the Oocyte Retrieval Set
4 Transvaginal Oocyte Retrieval Technique
5 Post Procedure Care
6 Complications
7 Other Routes for Oocyte Retrieval
8 Dos and Dont’s
9 Debatable Issues
10 Quality Assurance and Performance
11 Conclusion
Embryo Transfer
1 Introduction
1.1 Embryo Transfer Techniques
2 Scheduling and Consents
2.1 Day of Transfer
2.2 No. of Embryos to be Transferred
2.3 Recommendations for the Limit to the Number of Embryos to Transfer
2.4 Grading of Embryos
2.5 Blastocyst Grading Istanbul Consensus
3 Trial Transfer and Difficulty Assessment
4 Embryo Transfer Procedure
4.1 Setup for ET OT or ET Suit
4.2 Setup for ET Embryology Laboratory
4.3 Equipment and Disposable
4.4 ET Catheter
4.5 ET Transfer Procedure
4.5.1 IVF Lab Procedure
4.6 Loading Techniques
4.7 Embryo Transfer Clinical Procedure
4.8 Remaining Embryos
Intrauterine Insemination
1 Follicle Monitoring and Timing of IUI
2 Sample Collection
3 Semen Analysis
4 Semen Processing
5 Procedure
6 Post Procedure Instructions
7 Types of IUI Catheters
8 Strategies to Overcome Difficulties in IUI
9 Double Vs. Single IUI
Sperm Retrieval Techniques
1 Introduction
2 Sperm Retrieval Techniques
2.1 Materials, Equipment, and Reagents
2.1.1 Operating Room
2.1.2 IVF Laboratory
2.1.3 Laboratory Setup
2.2 PESA
2.3 MESA
2.4 TESA
2.5 Testicular Sperm Extraction (TESE)
2.6 Micro-TESE
3 Surgical Complications
4 Results
Part III: Gametes: Sperm and Oocyte
Oocyte Morphology
1 Granularity in Cytoplasm
3 Cytoplasmic Inclusion
4 Vacoules
5 Shape
6 Zona Pellucida Abnormalities
7 Perivitteline Space Abnormalities
8 Polar Body
Semen Analysis
Human Sperm Morphology
1 Slide Preparation
2 Normal Spermatozoa
2.1 Normal Spermatozoa
2.2 Abnormal Spermatozoa
3 Strict Criteria
3.1 Head Defects
3.1.1 Large Head
3.1.2 Microcephaly/Small Sperm Head
3.1.3 Amorphous Head
3.1.4 Tapered Head
3.1.5 Pyriform Head: (Fig. 7)
3.1.6 Small Acrosomal Area
3.2 Neck/Mid-Piece Defect
3.2.1 Bent Mid-Piece
3.2.2 Over- Thick Mid-Piece
3.2.3 Cytoplasmic Droplet
3.3 Tail Defect
3.3.1 Coiled Tail
3.3.2 Tail-Tip Coiling
3.3.3 Double Tail
Assessment of Sperm DNA Damage
Retrograde Ejaculation
1 Part 1. Introduction
2 Part 2. Signs of Retrograde and Clinical Investigation
3 Part 3. Laboratory Confirmation of Retrograde Ejaculation
3.1 Patient Preparation
3.2 Laboratory Diagnosis (Fig. 3)
4 Part 4. Preparation of Retrograde Samples for ART
Part IV: Embryo
Embryo Morphology and Embryoscopy
1 Cleavage Stage Embryo
2 Compaction
3 Blastocyst
4 Morphology Vs. Morphokinetics
The Blastocyst
1 Expansion Grades of a Blastocyst
2 Inner Cell Mass and Trophectoderm
3 Blastocyst with Excluded Cells or “Fragmentation”
4 Abnormally Shaped Blastocyst
5 Site of Hatching of Blastocyst
6 Expansion and Contraction of a Blastocyst
7 Cytoplasmic Strings in Blastocyst
Part V: Embryology Procedures
Semen Preparation Techniques
1 Introduction
2 Collection of Semen
3 Semen Analysis Before Preparation
4 Sperm Preparation Methods
4.1 Swim-Up
4.1.1 Layering and Swim-Up
Media Used
4.2 Density Gradient Centrifugation
4.2.1 Procedure
4.3 Magnetic Activated Cell Sorting
4.4 Microfluidic Sperm Sorting (MFSS)
4.4.1 Procedure
5 Conclusion
1 Sperm Cryopreservation
1.1 Principle
2 Semen Collection and Preparation of Sperm for Cryopreservation (Fig. 1)
3 Cryopreservation Technique
3.1 Slow Freezing
3.2 Rapid Freezing [4]
3.3 Thawing (Fig. 5)
4 Oocyte and Embryo Cryopreservation
4.1 Introduction
4.2 Principle
4.3 Steps of Vitrification
4.3.1 Virtification Steps
4.3.2 Oocyte in Equilibrium Medium
4.3.3 Oocyte in Vitrification Medium
4.3.4 Oocyte Loading
4.3.5 Four Cell Stage in Equilibrium Medium
4.3.6 Blastocyst Stage in Equilibrium Medium
4.3.7 Blastocyst Loading
4.3.8 Liquid Nitrogen Storage Tank
ICSI and Micromanipulation
1 Introduction
2 Equipment
3 Setting Up of a Micromanipulator System
4 Preparation of Gametes for ICSI
4.1 Sperm Preparation
4.2 Oocyte Preparation
5 Micromanipulation Procedures
5.1 ICSI
5.2 Assisted Hatching
5.3 Embryo Biopsy
6 Conclusion
Preimplantation Genetic Testing (PGT)
1 Introduction
2 Embryo Biopsy
2.1 PGT-A
2.2 PGT-SR
2.3 PGT-M
3 Future Perspectives
Preimplantation Genetic Testing for Aneuploidy (PGT-A): Lab Aspects
1 State-of-Art Lab Facility for Embryo Biopsy
2 LASER: Laser Is an Important Tool Required to Enable an Embryo Biopsy
2.1 New Lasers
2.2 Dynamic Laser
2.3 Static Laser
3 Micromanipulator
4 Diagrammatic Representation of Events in a PGT-A
5 Assisted Hatching for Embryo Biopsy
5.1 Chemical Hatching
5.2 Mechanical Hatching
5.3 Laser-Assisted Hatching
6 Various Stages of Biopsies
6.1 Polar Body Biopsy
6.2 Cleavage Stage Biopsy
6.2.1 Advantages of Cleavage Stage Biopsy
6.2.2 Limitations of Cleavage Stage Biopsy
6.3 Morula Stage Biopsy
6.4 Blastocyst Biopsy
6.4.1 Advantages of TE Biopsy
6.4.2 Limitations of TE Biopsy
7 Timing for Laser-Assisted Hatching (LAH) for Blastocyst Biopsy: Day-3 Vs. Day-5
8 Mosaicism
8.1 Recommendations for the Laboratory (if Reporting Mosaic Aneuploidies)
8.2 Recommendations for the Clinician
9 Grade of Embryo and Correlation with Aneuploidy
10 When to Freeze After Biopsy?
10.1 Cryopreservation of Embryos

Citation preview

Atlas of Assisted Reproductive Technologies Surveen Ghumman Editor


Atlas of Assisted Reproductive Technologies

Surveen Ghumman Editor

Atlas of Assisted Reproductive Technologies

Editor Surveen Ghumman IVF and Reproductive Medicine MAX Superspeciality Hospitals New Delhi, India

ISBN 978-981-99-0018-3    ISBN 978-981-99-0020-6 (eBook) © Springer Nature Singapore Pte Ltd. 2023 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore


Atlas of Assisted Reproductive Technologies is a much needed book in this field where many of the procedures are done under the microscope visible only to the one who is directly involved. An atlas becomes a means of sharing experience in the IVF laboratory. The initial pages cover the clinical aspect and the setup of an IVF laboratory and equipment. The chapter on ultrasound, which is one of the most important imaging techniques required in ART, is detailed with follicular monitoring and Doppler studies and its implications in ART.  Pictures to show oocyte retrieval, embryo transfer, and IUI are self-explanatory about the methods and the difficulties that can occur. The chapter on sperm retrieval techniques is exhaustive and shows detailed techniques of TESA and Microtese. There are chapters dedicated to laboratory work with respect to the sperm, oocyte, and embryo. Aspects of semen analysis, sperm morphology, sperm DNA damage assessment, and sperm preparation methods have been covered in detail through pictures. Retrograde ejaculation has been discussed with the clinical and laboratory aspects in mind. There is no better way of depicting oocyte and embryo morphology and grading than pictures and this is well represented in the Atlas. The chapters on cryopreservation and micromanipulation techniques touch all the finer details which need to be known. The embryo biopsy and PGS have been shown explicitly through figures for better understanding on the subject. This book would help in understanding both the basics and the complexities of the subject. It has researched hands-on knowledge related to the subject and would road map step-by-step management of both clinical and laboratory aspects of ART. New Delhi, India

Surveen Ghumman



I would like to express my deepest gratitude to all our eminent authors for their valuable contributions. Their best efforts have helped to give the book its shape and content it. I am grateful to my patients for giving me the opportunity to have immense practical experience to put this work into print in the form of this book. A special acknowledgement to Springer Publishing for their support, patience, and encouragement. I would like to appreciate the constant support I get from my family in all my academic endeavors and specially for providing motivation and encouragement to accomplish this endeavor to fruition.



Part I ART Laboratory and Equipment  Up of an IVF Lab��������������������������������������������������������������������������������������    3 Set Sudha Prasad and Saumya Prasad Equipment for Micromanipulation����������������������������������������������������������������   19 Kuldeep Jain and Maansi Jain Part II Clinical Procedures  Ultrasound Imaging of the Ovary in Infertility��������������������������������������������   29 Sonal Panchal and Chaitanya Nagori Oocyte Retrieval����������������������������������������������������������������������������������������������   43 Nikita Naredi Embryo Transfer����������������������������������������������������������������������������������������������   61 Umesh N. Jindal, Sanjeev Maheshwari, Manisha Jain, and Shefali Agnihotri Intrauterine Insemination ������������������������������������������������������������������������������   89 Surveen Ghumman and Parul Aggarwal Sperm Retrieval Techniques ��������������������������������������������������������������������������  101 Fabio Coltro Neto, Bárbara Ferrarezi, and Sandro C. Esteves Part III Gametes: Sperm and Oocyte Oocyte Morphology ����������������������������������������������������������������������������������������  123 Dhannya Binoy, Alex C. Varghese, Sreesha Viswam, and Raiza Ashraf Semen Analysis ������������������������������������������������������������������������������������������������  167 Stuart Benjamin John Dawe-Long Human Sperm Morphology����������������������������������������������������������������������������  179 Sandesh K. Patel ix



 Assessment of Sperm DNA Damage��������������������������������������������������������������  189 Guruprasad Kalthur, Sandhya Kumari, and Satish Kumar Adiga Retrograde Ejaculation ����������������������������������������������������������������������������������  195 Stuart Benjamin John Dawe-Long Part IV Embryo  Embryo Morphology and Embryoscopy��������������������������������������������������������  205 Keshav Malhotra, Jaideep Malhotra, Narendra Malhotra, and Neharika Malhotra The Blastocyst��������������������������������������������������������������������������������������������������  213 Gaurav Majumdar Part V Embryology Procedures Semen Preparation Techniques����������������������������������������������������������������������  227 Kanad Dev Nayar, Gaurav Kant, and Shweta Gupta Cryopreservation����������������������������������������������������������������������������������������������  253 Sandesh K. Patel and Sonu T. Lukose ICSI and Micromanipulation��������������������������������������������������������������������������  263 Sujatha Ramakrishnan and Yasoda Kiran Preimplantation Genetic Testing (PGT)��������������������������������������������������������  281 Carmen Morales, Daniel González, Rosa Bautista-Llacer, and Esther Velilla Preimplantation Genetic Testing for Aneuploidy (PGT-A): Lab Aspects����������������������������������������������������������������  307 Sameer Singh Thakur, Krishna Mantravadi, and Durga G. Rao

Part I

ART Laboratory and Equipment

Set Up of an IVF Lab Sudha Prasad and Saumya Prasad

The ART laboratory requires a sterile, stable, and non-toxic environment. Therefore, it is very critical to setup an optimum ART laboratory. The layout of the ART lab is based on the anticipated caseload, the budget available, and future plans of expansion (Fig. 1). Location of the laboratory should ideally be first floor since there will be less disturbance. Place should be checked for any damp area or water seepage.

VIP Lounge

Cons - 1

Cons - 4

Cons - 2

Cons - 5

Cons - 3

Cons - 6

Library & Data Entry

Scientific Director’s Room

USG Medical Director’s Chamber


Reception area


Admin Area (Open)

UPS Room

Andrology Lab

Sample Collection Room

Recovery Area

Sperm Lab

Surgeon’s Waiting Area

Training Lab

OT Storage Area

Embryology Lab Molecular Genetics Lab

Coys Facility

Nursing Station Cum Injection Room

Consulting Room


Conference Room

Semen Collection Room


Freezing Room Holistic Therapy




Fig. 1  IVF lab layout

S. Prasad · S. Prasad (*) Matritava Advanced IVF and Training Center, New Delhi, India © Springer Nature Singapore Pte Ltd. 2023 S. Ghumman (ed.), Atlas of Assisted Reproductive Technologies,



S. Prasad and S. Prasad

1 Non-Sterile Area 1. A reception and waiting room for patients (Fig. 2). 2. Room for clinical examination: A room with privacy for interviewing and examining patients. It must have an examination table, gynecological examinations, and ultrasound machine (Fig. 3). 3. Store room: Store room is required for storage of sterile items (media, needles, catheters, petri dish) and non-sterile materials under refrigerated and non-­ refrigerated conditions (Fig. 4). 4. Record room: A room to record essential details of patients (Fig. 5).

Fig. 2  Reception room

Fig. 3  Examination and ultrasound room

Set Up of an IVF Lab

Fig. 4  Store room Fig. 5  Record room



S. Prasad and S. Prasad

5. Autoclave room: Autoclave room is used for sterilizing and autoclaving all surgical items as well as clothes for patients (Fig. 6). 6. Semen collection room: It should have privacy and appropriate environment, located in a secluded area close to the laboratory. It should have instructions for semen collection, sterile and non-toxic container, washbasin, soap and clean towels, and an attached toilet (Fig. 7). Fig. 6  Autoclave room

Fig. 7 Semen collection room

Set Up of an IVF Lab


7. Semen processing laboratory: it should be close to the semen collection room (Fig. 8). Equipments for semen processing (Fig. 9):

(a) Laminar air flow. (b) Centrifuge machine.

Fig. 8 Semen processing lab

Fig. 9  Laminar flow with microscope and heating block


S. Prasad and S. Prasad

Fig. 10  IUI room

(c) Dry incubator. (d) Binocular or phase contrast microscope. (e) Facilities for microscopic examination. (f) Refrigerator. (g) Heating block. (h) Sperm counting chambers, such as Makler Chamber, Neubaur Chamber.

8. Clean room for IUI: The IUI room should have an appropriate table for conducting an IUI (Fig. 10).

2 Sterile Area Entry strictly controlled with initially an • Area for changing into sterile garments. • Scrub station. The sterile area must be air conditioned where fresh air is filtered through an approved and appropriate filter system that is circulated at ambient temperature (22–25 °C). 1. Operation theatre: Operation theatre should have provision for endoscopy and transvaginal ovum pick up. It should be equipped with emergency resuscitative procedures.

Set Up of an IVF Lab


2. Embryo transfer room: It is a room for intrauterine transfer of embryo under ultrasound guidance (Fig. 11). 3. Embryology Laboratory (Fig. 12): Mimicking in vivo atmosphere is extremely important in terms of temperature, humidity and air quality, to optimize fertilization, cleavage, blastulation, implantation, and pregnancy rates [1]. Fig. 11  Operation theatre and embryo transfer room with ultrasound machine

Fig. 12  IVF lab


S. Prasad and S. Prasad

3 Infrastructure of the IVF Laboratory 1. Walls and Ceiling: (Fig. 13)

(a) Walls and partitions should be made of non-porous inert material which provides an inert (low VOC), hypoallergenic environment. (b) The ceiling must be composed of a contiguous, solid material not tiles, and the need for any access panels must be minimized.

2. Paints

(a) Paints which emit VOCs (amine) and take several weeks to cure should be avoided. (b) Emission testing on samples is required since amine catalysts can be very persistent. (c) No paint containing formaldehyde, acetaldehyde, isocyanates, reactive amines, phenols, or soluble VOCs should be used.

3. Floors (Fig. 14)

(a) Large vitrified tiles/vinyl sheets or treated marble should be preferred. (b) Floor should be impervious, sealed with minimum joints with curved edges for easy cleaning.

4. Water supply (Fig. 15)

(a) A scrub station with water supply which is from separate stainless steel tanks for lab not from common storage tanks of building.

Fig. 13  Walls and ceiling

Set Up of an IVF Lab


Fig. 14 Floor

Fig. 15  Scrub station and water supply

(b) Sinks and drains should be outside lab. Ducts/pipes should be hidden between wall and panels or covered.

5. Gas supply (Fig. 16)

(a) Inert stainless steel tubing or Teflon-coated tubing with medical gasses (CO2) recommended for incubators. Copper to be avoided since it is prone to oxidation.


S. Prasad and S. Prasad

Fig. 16  Gas supply

Fig. 17 Door

6. Doors (Fig. 17)

(a) They should be coated with steel and have pass-through windows.

7. Furniture (Fig. 18)

(a) Wooden furniture should be avoided as it releases VOCs.

Set Up of an IVF Lab


Fig. 18 Furniture

(b) Painted surfaces can release VOCs and should be avoided. If completely unavoidable, then it should be high density and termite proof. (c) Cabinets should be powder-coated metal or stainless steel. (d) Stainless steel/Aluminum furniture is preferred. Placement of incubators, gamete-handling areas, workstations should be such that embryologist should be able to finish one complete procedure without moving more than 3 m in any direction

8. Light (Fig. 19)

(a) The lighting should be within sealed lighting units in order to help reduce airborne particles and should be dimmable. (b) Direct sunlight, UV light, and fluorescent light are detrimental to embryos [2]. (c) Visible blue (wavelength of 445–500 nm) has been shown to impact blastocyst formation. (d) Light damages by increasing ROS/free radicals (embryotoxic).

9. Electricity (Fig. 20)

(a) Electricity is critical with zero tolerance to interruption. Backup must be provided within seconds. (b) Power point sockets are to be placed at regular distances. (c) No window AC to be installed.


S. Prasad and S. Prasad

Fig. 19 Lights

Fig. 20 Electricity

10. HVAC (heating, ventilation,and air conditioning system) (Fig. 21).

(a) Helps to deliver clean air to room (b) Removes contaminants from sources in room. (c) Pressurizes room. (d) Controls temperature and humidity. (e) Should have HEPA filters.

Set Up of an IVF Lab


Fig. 21 HVAC

4 HEPA Filters (High-Efficiency Particulate Air) (Fig. 22) 1. It removes 99.97% of airborne particles more than 0.3 μm size. 2. It does not remove VOC.  Only activated carbon/KMnO4 can remove VOCs. Hence, a carbon filter is used.


S. Prasad and S. Prasad

Fig. 22 HEPA

5 Air Handling Unit (AHU) (Fig. 23) Based on the external air quality, the ratio of external flow and internal recirculation is adjusted.

Fig. 23 AHU















Set Up of an IVF Lab 17


S. Prasad and S. Prasad

References 1. Swain JE. The culture environment in the IVF laboratory: impact of pH and buffer capacity on gamete and embryo quality. Reprod Biomed Online. 2010;21(1):6–16. 2. Revised Guidelines for Good Practice in IVF Laboratories. 2015. https://www.eshre. eu/Guidelines-­a nd-­L egal/Guidelines/Revised-­g uidelines-­f or-­g ood-­p ractice-­i n-­I VF-­ laboratories-­(2015).aspx.

Equipment for Micromanipulation Kuldeep Jain and Maansi Jain

1 ICSI Instrumentation 1.1 Main Components 1. Inverted microscope (Fig.  1). Inverted microscope equipped with Hoffmann modulation contrast or differential interference contrast and 4×, 10×, 20×, and 40× with heated stage (Table 1). 2. Micromanipulator (Figs. 1 and 2). Micromanipulator consists of 2 units (one for moving the holding pipette and another for injection needle to transfer the sperm). Accessories

(a) Adaptor for inverted microscope. (b) Air/Oil microinjector for holding the oocyte. (c) Oil microinjector for transferring the sperm. (d) Injection pipette. (e) Holding pipette. (f) CCD camera with high-resolution screen.

K. Jain (*) · M. Jain KJIVF and Laparoscopic Centre, New Delhi, Delhi, India e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2023 S. Ghumman (ed.), Atlas of Assisted Reproductive Technologies,



K. Jain and M. Jain

Fig. 1  Olympus inverted microscope IX71 with Hofmann’s modulation

1.2 Types of Micromanipulators: Micromanipulators can be of three types 1. Manual 2. Hydraulic 3. Electronic Commonly used micromanipulators are

(a) Narishige manipulator (Figs. 3 and 4) (Table 2) (b) Integra-RI (Table 2) (c) Eppendorf - Fully motorized


Equipment for Micromanipulation Table 1  Microscope components and their function Parts of microscope (Fig. 2) 1. Diaphragm/iris/Apperture

2. Modulation control system 3. Condenser height adjuster 4. Eye pieces with focus control with adjustable width between the eyepieces 5. Light controls 6. Hoffmann modulation component:   (a) Circular polarising filter with rotation   (b) Condensor sensoring screws 7. Carousel and slit aperture filters 8. Condensor 9. Kohler illumination setup

Fig. 2 Hofmann modulation with heating stage and light assembly

Function • Control width of light • Reduce stray light or noise in the image in the view Makes oocyte which are transparent more visible by adding contrast to the viewable image For optimum setup by controlling the height of condenser by wheels of the adjustor To see To adjust intensity/brightness Modulating contrast of the image To center the condensor Matched with the objectives To converge incoming light to a cone to focus incoming light to the object being viewed. Uniform contrast better illumination and resolution

22 Fig. 3 Complete assembly: Narishige micromanipulator ON 3 with Olympus inverted microscope IX 71, front view

Fig. 4 Narishige micromanipulator ON 3: with Olympus inverted microscope, side view

K. Jain and M. Jain


Equipment for Micromanipulation Table 2  Comparison between Narishige and RI-Integra systems Features Controls Choice of air and oil syringes Touch screen controls Motion-triggered light

Narishige Precise and intuitive control Yes

RI–Integra Same Yes

No No



Versatility/ user-friendly Colour touch screen Fully customizable USB connectivity Heating system Microscope compatibility Micropipette compatibility


Yes Yes, helps to improve visibility during change Yes, to track samples or procedures, on-screen stopwatch Yes

No Yes No Customizable Can be connected to all leading microscopes Compatible to all leading pipettes present

Yes Yes Yes, RI viewer software available Customizable Can be connected to all leading microscopes Compatible to all leading pipettes present

1.3 Function of Manipulators Converts macroscale (cm) movement to microscale (μm) without compromising viability of gametes/embryo 1.3.1 Hydraulic Micromanipulator Components and Their Function Consists of 2 units: 1. Control unit: magnetic stand fixed on metal plate by magnetic stick for easy movement of joystick which is in two dimensions. There are three rotating knobs, one at the end of the joystick and two above the joysticks (Figs. 3, 4, 5 and 6). Simultaneous movement in three dimensions using joystick and protruding knob is done. The movement ratio adjustment ring is used to control amplitude of joystick movement along with tension adjustment ring [1] 2. Drive unit: It is mounted on a course manipulator and connected to control unit by three tubings filled with oil. Movement of drive unit is transferred to a microtool [2]. Movement of knob and joysticks creates pressure which is transferred to oil and to drive unit proportional to amplitude and speed of joystick and knob. This is achieved mechanically, electronically, or hydraulically. It helps in operating the holding pipette and injecting pipette (Figs. 5 and 7). Parts of microscope and its function are given in Table 1. Narishige Vs. RI-Integra: Comparison (Table 2).


K. Jain and M. Jain

Fig. 5  Joystick with injector—Semi-motorized

Fig. 6  Heating stage and Joystick controls

1.4  Eppendrof Micromanipulator: Fully Motorized • Unique dual speed joystick for precise, instantaneous control, and positioning using two different speed modes. • Ergonomically shaped control panel for fatigue-free work. • Optimized user interface for various applications simplifies work procedures. • Simple and quick capillary and sample change using automated home function. • Selection and programming of additional functions (e.g. storage of up to 5 positions, limit, Y-off).

Equipment for Micromanipulation


Fig. 7  Holding and injection pipettes

• Comfortable, individual speed adjustment. • The easily adjustable angle of the holding and injection capillaries can be set from 0° to 90°. • Easy adjustable angle from 0° to 90°. • Plug-and-play system with scale to allow for easy installation. • Swivel joint to allow for easy capillary and sample exchange.

References 1. Timson J, McDermott H. Instrumentation and preparation for micromanipulation. Baillieres Clin Obstet Gynaecol. 1994;8(1):13–41. 2. Vratny M.  Equipping a micromanipulation workstation. Am Biotechnol Lab. 1990;8(5): 36, 38–41.

Part II

Clinical Procedures

Ultrasound Imaging of the Ovary in Infertility Sonal Panchal and Chaitanya Nagori

Ovulation and implantation are the two major events of fertility in female. Ovaries, a paired organ, are responsible for ovulation as well as for production of steroids like oestrogen and progesterone to support the preparation of the endometrium and process of implantation. Ovaries are situated on either side of the uterus in ovarian fossa, suspended by utero-ovarian ligament and held in the broad ligament. These are supported from the pelvic wall by the suspensory ligament. Lateral to the ovary is the external iliac vessels, as seen on longitudinal plane and on transverse plane, it is between external iliac artery laterally and internal iliac artery medially. • Surface: The surface layer of the ovary is formed by simple cuboidal epithelium, known as germinal epithelium. • Cortex: The cortex (outer part) of the ovary is largely comprised of a connective tissue stroma. It supports thousands of follicles. Each primordial follicle contains an oocyte surrounded by a single layer of follicular cells. • Medulla: The medulla (inner part) is composed of supporting stroma and contains a rich neurovascular network which enters the hilum of ovary from the mesovarium. On ultrasound, ovaries appear as usually oval soft tissue organs with hypo to isoechoic stroma and several round anechoic areas, follicles distributed throughout the ovary. Its volume may vary from 3.0 cc to 6.6 cc and is calculated as D1 × D2 × D3 × 0.523. (D1: maximum long diameter on the longest section of the ovary). This is found by rotating the probe with ovary in vision to find the long axis and then scrolling across to find the longest diameter plane. D2: Antero-posterior diameter: longest diameter perpendicular to the long diameter on the same section. D3: width: Longest side to side diameter, on the section achieved by 90° rotation from the longest plane (Fig. 1).

S. Panchal (*) · C. Nagori Dr. Nagori’s Institute for Infertility and IVF, Ahmedabad, India © Springer Nature Singapore Pte Ltd. 2023 S. Ghumman (ed.), Atlas of Assisted Reproductive Technologies,



S. Panchal and C. Nagori

Fig. 1  B mode ultrasound image of the ovary in longitudinal and transverse sections to measure the three orthogonal ovarian diameters to calculate volume

The ovarian size and structure are both dynamic due to the changes occurring in the ovarian morphology continuously as a result of recruitment of the antral follicles, their growth to maturity and rupture for ovulation resulting finally into a cystic structure with hemorrhage inside when it is named as corpus luteum. Out of several antral follicles recruited, one or two may attain maturity. The antral follicles that were recruited but do not reach maturity undergo atresia. Corpus luteum also undergoes regression and disappears in the course of time during the menstrual cycle. From primordial follicles to primary antral follicles and secondary antral follicles is a long phase (about 150 days) of growth. Secondary antral follicles (2 mm) are the first follicular structures that may be visualized on ultrasound (US). Antral follicles can be counted on B mode but when multiple, 3D ultrasound with SonoAVC would be a more accurate method as it colour codes the follicles and measures the size of the follicles (Fig. 2a, b). Beyond this, the follicular growth is also fast and feasible to monitor. Once a follicle reaches a diameter of >9 mm, it is a dominant follicle and beyond that it has a steady growth rate of 2–3 mm a day [1]. When the follicle reaches a diameter of 18 mm, it is considered to be mature. It may show a perifollicular halo and a cumulus has a tiny solid projection (Fig. 3a). Cumulus can be better demonstrated on 3D ultrasound (Fig. 3b). Feichtinger et al. in their study have shown presence of cumulus in follicles >15 mm by 3D US [2]. However, this is just anatomical maturity of the follicle. Doppler assessment is required to assess the functional maturity of the follicle. Increase in perifollicular

Ultrasound Imaging of the Ovary in Infertility




Fig. 2 (a) 3D ultrasound image of the ovary with sono AVC showing colour coded antral follicles. (b) Sono AVC worksheet showing x, y and z diameters and volume of each follicle, mentioned against its colour code



Fig. 3  Mature follicle on B mode showing a peri-follicular halo and a tiny solid projection cumulus(arrow)in figure (a) and 3D ultrasound image of the same follicle showing cumulus(arrow) in figure (b)

vascularity of dominant follicle in theca layer starts developing as early as eighth day of the cycle. Fall in perifollicular resistance index (RI) starts 2 days before ovulation, reaches nadir at ovulation, remains low for 4 days and then with gradual rise reaches 0.5 in mid luteal phase [3]. The follicle is known to be functionally mature when the blood vessels cover three-fourth of the follicular circumference, and these vessels have (RI) of ≤0.48 and a peak systolic velocity (PSV) of >10 cm/s (Fig. 4a, b). Planning triggers when these perifollicular vascular parameters are achieved which optimizes the oocyte yield in patients on in vitro fertilization (IVF) treatment and improves the conception rate significantly in patients on intrauterine insemination (IUI) treatment [4]. Follicular vascularity is an indirect assessment of ovum oxygenation. Follicles with more uniform perifollicular vascular network are more likely to produce pregnancy [5]. While oocytes from severely hypoxic follicles have high frequency of abnormalities of organization of chromosomes on metaphase spindle and may lead to



S. Panchal and C. Nagori


Fig. 4  Perifollicular vascularity seen on colour Doppler in figure (a) and figure (b) showing spectral Doppler image of the same follicle with low resistance flow

Fig. 5  Multi-layered endometrium with endometrial flows seen on HD flow Doppler

segregation disorders and catastrophic mosaics in embryo [6]. As the LH surge starts, after the initial slow rise in LH level, there is a steep upward rise in the LH levels, and this leads to steep rise in the perifollicular PSV. Rising PSV in the perifollicular vessels indicates impending ovulation. The progressing follicular maturity leads to endometrial growth. It is the high oestrogen levels that lead to the multi-layered morphological pattern of the endometrium and also endometrial vascularity (Fig. 5). After ovulation rupture of the follicle, the corpus luteum appears like a thick walled cystic structure in the ovary with internal echogenecities. These echogenecities are lace-like or fishnet-like with fibrin strands due to the haemorrhage occurring in the follicle as a result of rupture and ovulation (Fig.  6a). Although corpus luteum may also sometimes show low level echoes or may appear isoechoic to the ovarian stroma (Fig. 6b). In these cases, only Doppler will help to identify the corpus luteum (Fig. 6c). Active corpus luteum shows the peripheral flow ring of the colour (Fig. 6d). A clear correlation between RI of corpus luteum and plasma progesterone levels has been seen in natural cycle [7]. It is essential therefore to assess the corpus luteal vascularity with Doppler in the midluteal phase. Normal corpus

Ultrasound Imaging of the Ovary in Infertility





d Fig. 6 (a) Thick walled corpus luteum with fishnet/lacelike echogenecities inside- corpus luteum. (b) Isoechoic corpus luteum. (c) Isoechoic corpus luteum, only recognised by the ring of vascularity seen by Doppler. (d) corpus luteum with fibrin strand echogenecities seen in the lumen on B mode and HD flow Doppler shows peripheral vascularity Fig. 7  On pulse Doppler, corpus luteal vessels show low resistance flow

luteum is covered by vascular ring with RI of ≤0.5 (Fig. 7). High RI of the corpus luteum and scanty vascularity indicate corpus luteal inadequacy and luteal phase defect. Luteinized unruptured follicle instead has thick hyperechoic walls with low level internal echogenecities and high resistance flow (Fig. 8).


S. Panchal and C. Nagori

Fig. 8  Thick hyperechoic walls of an intra-ovarian cystic lesion with low level internal echogenecities suggestive of luteinized unruptured follicle

Fig. 9  Intraovarian cystic lesion on B mode ultrasound with fishnet internal echogenecities, in absence of blood flows suggest haemorrhagic cyst

Hemorrhagic cyst shares the same B mode image as the corpus luteum. But as a rule, it shows no vascularity. This is because it is non-functional. It is a residual non-­ functional corpus luteum (Fig. 9). Endometriomas are commonly found in patients with infertility. These appear as thick-walled cystic intra-ovarian lesions with internal echogenecities. Internal echogenecities may be ground glass or lacelike (Fig. 10). There may be fluid level seen separating the fresh blood and debris (echogenic fluid) posteriorly from serum (anechoic fluid) anteriorly [8] (Fig. 11). Hyperechoic flecks in the walls of endometrioma are supposed to be haemosiderin deposits [9] (Fig. 12). With large endometriomas bilaterally, the ovaries may be displaced posterior to the uterus and may even get adherent to each other Kissing ovaries (Fig.  13). Adhesions and pain on probe pressure are supportive signs of endometriomas. On Doppler, these show short-coursed vessels (Fig. 14) which on 3D power Doppler typically gives a bird’s nest appearance [10].

Ultrasound Imaging of the Ovary in Infertility


Fig. 10  Intraovarian cystic lesion with ground glass internal echogenicity and thick shaggy walls on B mode ultrasound suggest the highest possibility of endometrioma

Fig. 11  Intraovarian cystic lesion with ground glass internal echogenicity and a vertical fluid level on B mode ultrasound suggest the highest possibility of endometrioma

Pregnancy leading to decidualization of the endometrioma or a rare malignant change may lead to development of solid projections and increased vascularity in these lesions. Dermoids may often be seen in the reproductive age group. On ultrasound, dermoid appears as cystic lesion with solid component with low level echoes with/ without hyperechoic lines or hyperechoic balls in the cystic component of the lesion (Fig. 15). The appearances vary according to the contents of the dermoids. Tooth-­ like hyperechoic lesions may be seen with acoustic shadowing (Fig. 16). Fluid level may be similar to that of endometrioma but with hyperechoic fluid (fat) anteriorly and hypoechoic fluid (the liquid part posteriorly, unlike endometriomas. The walls of the dermoid and solid component (Rokistansky’s tubercles) are typically avascular. Hyperechoic mass may be seen, the posterior margin of which is not identified: a tip of the iceberg sign.


S. Panchal and C. Nagori

Fig. 12  Hyperechoic flecks in the walls of the intraovarian cystic lesion with thick walls and ground glass echogenicity suggest heamosiderin deposits in endometrioma

Fig. 13  Kissing ovaries seen on B mode ultrasound image

Ultrasound Imaging of the Ovary in Infertility


Fig. 14  Short coursed vessels of endometrioma seen on power Doppler ultrasound

Fig. 15  B mode ultrasound image of an intraovarian cystic lesion with multiple hyperechoic lines and hypoechoic contents suggesting a dermoid



S. Panchal and C. Nagori


Fig. 16 (a) B mode ultrasound image of an intraovarian cystic lesion with hyperechoic lines and dots (may be due to hair) and tooth like hyperechoic areas (arrow) with posterior acoustic shadowing typical of a dermoid. (b) Snowball like hyperechoic intraovarian lesion with ill-defined posterior margins suggesting a dermoid

Fig. 17  Anechoic cystic lesion with no internal echogenicity, no solid projections or papillarities, in the ovary-simple cyst

Simple cysts (Fig. 17) in the ovaries are thin walled with anechoic contents and show no vascularity on Doppler. These remain usually static for more than 4–6 weeks. Fibromas (Fig. 18) are solid benign tumours of the ovary and are often an incidental finding. These are solid round/oval lesions, with whorled pattern (like fibroid in the uterus) and isoechoic to ovarian stroma. On Doppler, these show peripheral vascularity. When large, fibromas may show heterogenous echogenicity and at times calcification. Fibroma may sometimes be associated with peritoneal, pleural, and pericardial effusion and thus is named “Meig’s Syndrome.” Epithelial tumours of the ovary may be serous, mucinous, or endometriod tumours. These have a variable and overlapping appearance. They are most commonly complex cystic lesions with cystic and solid components and septae, and their histopathological nature can only be confirmed on biopsy. However, the

Ultrasound Imaging of the Ovary in Infertility




Fig. 18 (a, b) Respectively B mode and power Doppler ultrasound image of a well-defined round hypoechoic solid mass with peripheral vascularity suggesting a fibroma



Fig. 19 (a) B mode ultrasound image of an intraovarian complex (cystic lesion with solid component) lesion. The solid component shows irregular margins and heterogeneous echogenicity, raising a suspicion of malignancy. (b) 3D power Doppler image with glass body rendering also shows the irregular margins of the solid component and also heterogeneously distributed vascularity, further increasing the possibility of the lesion being malignant

heterogenous echogenicity, irregular shape of the solid components, thick septae, heterogenously distributed increased vascular density, and irregular vascular diameters are the features that favour the possibility of malignancy (Fig. 19). Malignant ovarian tumours are not common in females of reproductive age group. Large ovaries (>10 cc) with multiple antral follicles (>20) may be suggestive of polycystic ovaries. The follicles may be arranged at the periphery or may be generally distributed (Fig.  20a, b). Polycystic ovaries are also known to have stromal abundance and increased vascularity. Both these can be best demonstrated by 3D ultrasound and 3D power Doppler (Fig. 20c, d). On ovulation induction, these ovaries have a risk to go into hyperstimulation and are seen as large ovaries with multiple follicles on ultrasound (Fig. 20e). Ultrasound, combination of transabdominal, and transvaginal are considered to be the modality of choice for ovarian assessment.



S. Panchal and C. Nagori


c Fig. 20 (a) B mode image of polycystic ovary with generalized distribution of follicles. (b) B mode image of polycystic ovary with generalized distribution of follicles. (c) 3D power Doppler volume of the ovary with volume histogram showing VI(vascularity index), FI (flow index) and VFI (vascularity flow index) -3D power Doppler quantitative vascular indices in the box. (d) 3D ultrasound volume of the ovary with threshold volume showing follicular volume as the ‘below threshold volume’ in the box inlaid in the image and stromal volume as ‘above threshold volume’ in the box. (e) 3D ultrasound volume of the hyper-stimulated ovary

Ultrasound Imaging of the Ovary in Infertility


e Fig. 20 (continued)



S. Panchal and C. Nagori

References 1. Hackeloer BJ, Fleming R, Robinson HP. Correlation of ultrasonic and endocrinological assessment of human follicular development. Am J Obstet Gynecol. 1979;135:122. 2. Kurjak A, Kupesic-Urek S.  Infertility. In: Kurjak A, editor. Transvaginal Color Doppler. Nashville: Parthenon Publishing; 1991. p. 33–8. 3. Coulam CB, Stern JJ, Soenksen DM, Britten S, Bustillo M. Comparison of pulsatility indexes on the day of oocyte retrieval and embryo transfer. Hum Reprod. 1995;10:82–4. 4. Vlaisavljevic V, Reljic M, Gavric LV, Zarula D, Sergent N.  Measurement of perifollicular blood flow of the dominant preovulatory follicle, using three dimensional power doppler. Ultrasound Obstet Gynecol. 2003;22(5):520–6. 5. Blerkom V, Antezak M, Schrader R. The developmental potential of human oocyte is related to the dissolved oxygen content of follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics. Hum Reprod. 1997;12(5):1047–55. 6. Feichtinger W. Transvaginal three dimensional imaging for evaluation and treatment of infertility. In: Merz E, editor. 3D ultrasound in obstetrics and gynecology. Philadelphia: Lippincott Williams & Wilkins; 1998. p. 37–43. 7. Glock JL, Brumsted JR.  Colour flow pulsed Doppler ultrasound in diagnosing luteal phase defect. Fertil Steril. 1995;64:500–4. 8. Asch E, Levine D.  Variations in appearance of endometriomas. J Ultrasound Med. 2007;26(8):993–1002. 9. Patel MD, Feldstern VA, Chen DC, et  al. Endometriomas: diagnostic performances of US. Radiology. 1999;210:739–45. 10. Raine-Fenning N, Jayaprakasan K, Deb S. Three-dimensional ultrasonographic characteristics of endometriomata. Ultrasound Obstet Gynecol. 2008;31:718–24.

Oocyte Retrieval Nikita Naredi

Oocyte retrieval (OR), or Ovum Pick Up (OPU), is a technique which forms an integral part of an in-vitro fertilisation cycle (IVF) for harvesting oocytes from the ovary of a woman, thus enabling fertilisation outside the body. Oocyte retrieval, which is now carried out transvaginally under ultrasound guidance, was once a real challenge when it had to be collected by laparotomy and/or by other laparoscopic techniques [1]. These techniques were not only difficult but unsuccessful in 50% cases. Introduction of the foot-controlled fixed aspiration pressure control and specially designed Teflon-lined aspiration needles with bevelled ends brought about a success rate of OPU to 60–80% [2, 3]. It was in 1984 at Strasbourg, France, when Pierre Dellenbach and colleagues revolutionised oocyte retrieval by carrying out Transvaginal Oocyte Retrieval (TVOR) under ultrasound guidance which is now the indispensable and standard method of harvesting oocytes [4].

1 Equipment and Consumables Required for Ovum Pick Up The following equipment/accessories for OPU should be mandatory • • • • •

Ultrasound machine with endovaginal probe (Fig. 1). Foot-controlled fixed oocyte aspiration pump (Fig. 2). Needle guide to be affixed on the endovaginal probe (Fig. 3). Speculum for cervical examination and to visualise any bleeding site. Oocyte Retrieval Set: A single-lumen 17- or 18-gauge needle (Fig. 4).

N. Naredi (*) Assisted Reproductive Technology Centre, Command Hospital, Pune, India © Springer Nature Singapore Pte Ltd. 2023 S. Ghumman (ed.), Atlas of Assisted Reproductive Technologies,



N. Naredi

Fig. 1 Ultrasound machine with endovaginal probe

Fig. 2 Foot-controlled fixed aspiration pump

• Test tube warmer and heating block (at 37 °C)/Mobile nest for transporting the tubes (Figs. 5 and 6). • IVF Grade Test Tubes (5 mL and 14 mL) (Fig. 7). • Culture medium for flushing the needle equilibrated at 37 °C (Fig. 8). Additional equipment and consumables that might be used during OPU should also be available in the procedure room, such as gauze pieces, non-powdered gloves, clamps, sponge holder, vaginal surgery equipment, including (absorbable) sutures.

Oocyte Retrieval


Fig. 3  Needle guide to be affixed to the endovaginal probe



Fig. 4 (a) Oocyte retrieval set: A single-lumen 17- or 18-gauge needle (packed). (b) Oocyte retrieval set: A single-lumen 17- or 18-gauge needle (opened)

Resuscitation equipment, reversal anaesthetic drugs, a prepared kit for anaphylactic shock treatment and oxygen should also be available in (the near proximity of) the procedure room.

46 Fig. 5  Test tube warmer and heating block at 37 °C

Fig. 6  Mobile nest for transporting the tubes

Fig. 7  IVF grade test tubes (5 mL &14 mL)

N. Naredi

Oocyte Retrieval


Fig. 8  Culture medium for flushing the needle equilibrated at 37 °C

2 Patient Preparation and Anaesthesia 1. A trolley with the necessary sterile instruments and accessories for patient preparation is made ready (Fig. 9). 2. The patient is instructed to void or empty the bladder completely before the procedure. 3. The patient is prepped and draped in the dorsal lithotomy position under intravenous sedation (Fig. 10). 4. Local part is prepared by thorough washing with lukewarm normal saline to eliminate any infection and contamination. Use of antiseptics may be toxic to the oocytes and are therefore not used in many centres.


N. Naredi

Fig. 9  A trolley with the necessary sterile instruments and accessories for patient preparation

Fig. 10  Patient prepped and draped in the dorsal lithotomy position under intravenous sedation

3 Preparation of the Ultrasound Machine, the Probe and the Oocyte Retrieval Set 1. The ultrasound machine having Micro Convex Endocavity Probe (5–10 MHz) is brought in the transvaginal mode setting (Fig. 11). 2. A metallic needle guide is attached over the probe in specified groove after covering the probe with the sterile probe cover (Fig. 12). 3. A small amount of conducting jelly is put at the tip of the probe before applying probe cover for better conduction and visibility. 4. A 17/18G, 35 cm, single lumen OPU needle of any make with echo tip marking for better tip orientation during OPU is inserted in the metallic needle guide (Figs. 13 and 14).

Oocyte Retrieval Fig. 11 Ultrasound machine having micro convex endocavitary probe (5–10 MHz) brought into the transvaginal mode setting

Fig. 12  A metallic needle guide attached over the probe with the sterile probe cover


50 Fig. 13  A 17/18G, 35 cm, single lumen OPU needle of any make with echo tip marking for better tip orientation during OPU

N. Naredi

Oocyte Retrieval


5. One end of the tubing is attached to the collection tube (14 mL tube) and other end is attached to the ovum aspiration pump. The needle is then flushed with some media before introducing it inside the patient. (It ensures proper aspiration of the ovum aspiration system as well as flushes out the needle.) (Figs.  15 and 16). 6. Pressure of the aspiration pump should be 100–120 mm of mercury (Fig. 17).

Fig. 14  OPU needle inserted in the metallic needle guide

Fig. 15  One end of the tubing attached to the collection tube (14  mL tube) and other end is attached to the ovum aspiration pump


N. Naredi

Fig. 16  The needle getting flushed with media before introducing it inside the patient

Fig. 17  Pressure of the aspiration pump to be 100–120 mm of mercury

4 Transvaginal Oocyte Retrieval Technique 1. After ensuring adequate anaesthesia, the needle is introduced into the pelvis through the vagina. 2. The vaginal probe should be fully opposed to the vaginal wall to properly visualise the target ovary. The amount of pressure on the vaginal wall must be per-

Oocyte Retrieval


sistent and sustained throughout the OPU procedure with one hand. The other hand should be left free to manipulate the needle for aspiration of the follicles. 3. The ovary should be lined up to the most accessible position on the screen and the follicle to be aspirated should be aligned in the largest diameter (Fig. 18). 4. Once the transducer is against the ovary (through the vaginal wall), the needle should be carefully inserted inside the follicle (Fig. 19). 5. Ideally the needle should be inserted in the middle of the ovary as it not only prevents the ovary from moving but also avoids the needle to cause injury to adjacent organs/vessels (Fig. 20). 6. The echogenic tip of the needle should be identified during all maneuvers. The needle guide seen on the monitor can facilitate safer insertion as it projects where the needle tip will progress. 7. The needle should be kept in the centre of the follicle during aspiration and observed for collapsing of the follicle walls around the tip. Fig. 18  The ovary lined up to the most accessible position on the screen and the follicle to be aspirated aligned in the largest diameter

Fig. 19  The needle should be inserted inside the follicle


N. Naredi

Fig. 20  The needle to be inserted in the middle of the ovary to prevent the ovary from moving and avoiding injury to adjacent organs/vessels

Fig. 21  The follicular fluid containing the oocyte/cumulus complex

8. The follicular fluid containing the oocyte/cumulus complex should be aspirated completely by application of suction which is evident by collapsing of the follicular wall (Fig. 21). 9. The needle can either be advanced into an adjacent follicle or withdrawn to the edge of the ovary and then moved to the next follicle; realignment is needed to advance into another follicle. Repetitive puncture through the vaginal wall should be minimised (Fig. 22). 10. Flushing of the needle should be done between the two ovaries to avoid or remove any blockage caused by blood clots (Fig. 23).

Oocyte Retrieval


Fig. 22  Realignment of the needle to advance into another follicle

Fig. 23  Flushing of the needle between the two ovaries to avoid or remove any blockage caused by blood clots

5 Post Procedure Care 1. At the end of the OPU, one must look at contours of both the ovaries and rule out any blood collection in the pelvis. 2. Local examination of the vagina is a must as vaginal bleeding can occur due to puncture: Vaginal bleeding in most of the instances is not troublesome and stops by applying pressure (Fig. 24). 3. Patient should be kept under observation for 2–3 h and oral feeds can be started after 3 h. 4. A check TVS must be carried out before the patient is sent home as TVOR is a day-care procedure. 5. Few patients may require single shot of analgesic for abdominal and vaginal pain.


N. Naredi

Fig. 24  Probe with vaginal bleeding due to puncture

6. In patients with hematoma, bleeding or infection after the OPU, antibiotic coverage is recommended. 7. Procedures/SOPs should be in place when severe complications occur, including hospital admission arrangements, specialist responsibility, and continued care.

6 Complications Overall, the incidence and severity of the reported complications after OPU are low [6–8]. Some of the reported complications include: • • • • • • • • •

Related to sedation, anaesthesia. Vaginal bleeding. Intra-abdominal/intra-peritoneal bleeding. Severe pain (requiring hospitalisation). Injury to pelvic structures. Pelvic infections. Pelvic abscess. Ovarian abscess. Ovarian Hyperstimulation Syndrome (a complication of Ovarian Stimulation and not oocyte retrieval per se) (Fig. 25). • Unsuccessful oocyte retrieval: “Empty follicle syndrome”: a clinical scenario wherein, despite the presence of ovarian follicles, no oocytes are retrieved at harvest.

Oocyte Retrieval


Fig. 25 Ovarian hyperstimulation syndrome (a complication of ovarian stimulation and not oocyte retrieval per se)

7 Other Routes for Oocyte Retrieval Ultrasound-guided transabdominal oocyte retrieval: Oocyte pick up is done transabdominally very rarely these days when the ovaries are not accessible vaginally, due to changes in the anatomy of pelvic organs which may be caused by biological variations or pelvic disease [9]. In these cases, the oocyte retrieval is performed by an alternative procedure, such as the transabdominal ultrasound-guided follicular aspiration [10, 11]. Laparoscopic Oocyte retrieval: It is again very rarely performed except for the same indications as the transabdominal oocyte retrieval: when the ovaries are transposed or enlarged above the pelvic brim and not accessible vaginally.

8 Dos and Dont’s 1. One has to be assured of the level of anaesthesia as needle puncture is very painful and low level of anaesthesia may cause patient’s undesirable movements and subsequently local injury by the needle. 2. Avoid contaminating the needle tip when introducing it in the vagina, by keeping the needle inside the needle guide until ready to pierce the vagina. 3. The needle tip markers must be observed at all times on the monitor as the needle is manoeuvred within the ovaries and into each follicle; the needle tip should never be advanced if not visible. 4. Avoid lateral movements of the needle to reduce the risk of vascular damage. 5. One should not move the transducer with the needle in the advanced position. 6. All follicles to be punctured, especially if there is a high risk of ovarian hyperstimulation syndrome (OHSS). 7. One can use colour-flow during aspiration because the position of the vessels also alters as the ovary moves after subsequent aspirations (Fig. 26).


N. Naredi

Fig. 26  Colour Doppler flow during aspiration to avoid vessel injury

8. A beneficial recommendation to the beginners is to put gain settings of ultrasound machine on higher side to brighten the capsule of the ovary, i.e., the limiting boundary of aspiration as it avoids inadvertent entry to the great vessels.

9 Debatable Issues 1. Need for prophylactic antibiotics: Most centres routinely use pre-retrieval intravenous antibiotic prophylaxis (e.g., cefoxitin, cefotaxime). However, some centres advocate only for high-risk patients (i.e., with a history of Pelvic Inflammatory Disease and/or endometriomas) [5, 6]. 2. Optimal Aspiration Pressure: At present, there is no conclusion on the optimal aspiration pressure level, and a variety of pressures between 100  mmHg and 200  mmHg are used. However, the optimal pressure is between 100 and 120 mmHg [12]. Most work reveals how low aspiration pressure resulted in a significantly greater oocyte yield, maturity, and fewer oocytes with an empty zona pellucida compared to high aspiration pressure [13]. 3. Flushing of the follicles to increase the oocyte number: there have been several reviews about flushing but follicle flushing has not been found to improve ART outcomes and requires longer procedure time [14].

10 Quality Assurance and Performance 1. Keeping clear and legible documentation regarding the OPU description with images and results in terms of number of oocytes obtained, difficulties encountered in the procedure. Information about the functionality, maintenance, and upgradation of the equipment should always be maintained as a good clinical practice.

Oocyte Retrieval


2. Forms should be available for admission and discharge, and also for reporting on the procedure. Quality assurance should analyse key clinical performance indicators, and it should be undertaken at least once per year in the IVF centre.

11 Conclusion The transvaginal ultrasound-guided follicle aspiration is a standard and preferable process for oocyte pick up as it is fast and minimally invasive compared to laparoscopy requiring minimal anaesthesia with fast recovery time. Furthermore, it is a highly safe, effective, and a reproducible tool for oocyte retrieval.

References 1. Lopata A, Johnston WIH, Leeton J, et  al. Collection of human oocytes by laparotomy and laparoscopy. Fertil Steril. 1974;25:1030–8. 2. Renou P, Trounson A, Wood C, et al. The collection of human oocytes for in-vitro fertilization. An instrument for maximizing oocyte recovery rate. Fertil Steril. 1981;35:409–12. 3. Wood C, Leeton J, Talbot M, et al. Technique for collecting mature human oocytes for in-vitro fertilization. Br J Obstet Gynaecol. 1981;88:756–60. 4. Dellenbach P, Nisand I, Moreau L, et al. Transvaginal, sonographically controlled ovarian follicle puncture for egg retrieval. Lancet. 1984;323:1467. 5. Srivastava P. Transvaginal oocyte retrieval in IVF: should we really be scared of the procedure? Gynecol Reprod Endocrinol. 2018;2(1):15–7. 6. ESHRE Working Group on Ultrasound in ART. Recommendations for good practice in ultrasound: oocyte pick-up. Hum Reprod Open. 2019;2019(4):hoz025. 7. Ludwig AK, Glawatz M, Griesinger G, Diedrich K, Ludwig M.  Perioperative and post-­ operative complications of transvaginal ultrasound-guided oocyte retrieval: prospective study of >1000 oocyte retrievals. Hum Reprod. 2006;21:3235–40. 8. Ozaltin S, Kumbasar S, Savan K.  Evaluation of complications developing during and after transvaginal ultrasound—guided oocyte retrieval. Ginekol Pol. 2018;89:1–6. 9. Seifer D, Collins R, Paushter D, George C, Quigley M. Follicular aspiration: a comparison of an ultrasonic endovaginal transducer with fixed needle guide and other retrieval methods. Fertil Steril. 1988;49:462–7. 10. Roman-Rodriguez CF, Weissbrot E, Hsu CD, Wong A, Siefert C, et al. Comparing transabdominal and transvaginal ultrasound-guided follicular aspiration: a risk assessment formula. Taiwan J Obstet Gynecol. 2015;54:693–9. 11. O'Shea RT, Forbes KL, Scopacasa L, Jones WR. Comparison of transabdominal and transvaginal pelvic ultrasonography for ovarian follicle assessment in in vitro fertilization. Gynecol Obstet Investig. 1988;26:52–5. 12. Panayotidis C. Dissertation: interventional ultrasound: standardisation of oocyte retrieval in assisted reproduction treatments. Cardiff: Postgraduate Medical School University of Cardiff, Cardiff University; 2017. 13. McQueen D, Boots CE, Jain T, Zhang JX, Robins J.  Impact of aspiration pressure during oocyte retrieval. Fertil Steril. 2018;110(4):E241–2. 14. Leung ASO, Dahan MH, Tan SL. Techniques and technology for human oocyte collection. Expert Rev Med Devices. 2016;13(8):701.

Embryo Transfer Umesh N. Jindal, Sanjeev Maheshwari, Manisha Jain, and Shefali Agnihotri

1 Introduction Embryo transfer (ET) is the final success-determining step in the process of achieving a pregnancy through Assisted Reproduction Techniques (ART). The learning curve is fairly long for this apparently simple procedure [1]. In addition to the embryo and endometrial receptivity, the success rates are operator dependent. The American Society for Assisted Reproduction (ASRM) has formulated guidelines and a template for standardized protocol for ET. It may be possible to improve success rate by 30% by using correct technique of ET [2]. Embryo transfer may be classified into various categories on the basis of the following: 1. Type of Embryos: Fresh or Frozen embryo transfers. Fresh embryo transfer may be postponed because of risk of OHSS, poor endometrium, high progesterone level, waiting for results of embryo biopsy or any other cause. 2. Day of Embryo transfer: Generally, it is done on day-3 or day-5. But few centers are still doing it on day-2. 3. Development stage of embryo: Embryo transfer may be done with cleavage stage embryo that is 4-cell or 8-cell embryo or blastocyst. 4. Number of embryos: It may be elective single embryo transfer (eSET) or more than one embryo transferred.

U. N. Jindal (*) · S. Maheshwari · M. Jain · S. Agnihotri Jindal IVF and Sant Memorial Nursing Home, Chandigarh, India © Springer Nature Singapore Pte Ltd. 2023 S. Ghumman (ed.), Atlas of Assisted Reproductive Technologies,


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5. Dual transfers: Generally, all embryos may be transferred in a single procedure but sometimes in repeated failure cases, embryo may be transferred in two procedures, first cleavage stage embryo on day-2 or day-3 followed by blastocyst transfer on day-5 or day-6. The embryos have to enter the endometrial cavity through the cervix as smoothly as it enters through the tubal ostium without any other disturbance in endometrial environment or iatrogenic damage to embryo during transfer. A poor-quality transfer technique can compromise the outcome even if best embryos are transferred in a very good endometrium. We will describe a pictorial stepwise description of ET technique followed at our centre at Chandigarh India (Fig. 1). The transfer involves deposition of embryos in the uterus at a point which has maximum receptivity (maximum implantation site, or MIP) without causing any trauma, bleeding, and disruption of endometrium or invoking any uterine contractions. Maximum implantation point has been described as the point in the middle of uterine cavity where two imaginary lines passing through the longitudinal axis of interstitial potions of the fallopian tubes intersect (Fig. 2). The embryos are released in the endometrium near the mid-cavity. This is typically about 5 cm from external cervical or and about 1–1.5 cm from the fundus. a









Fig. 1 (a) Diagrammatic anatomy of uterus, ovary, and fallopian tubes. (b) Diagrammatic procedure of embryo transfer

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Fig. 2 Maximum implantation site. (a) Diagrammatic representation (b) Ultrasound representation Fig. 3  Direct transfer technique

Ultrasound probe


Endometrial Cavity

Transferred embryo

1.1 Embryo Transfer Techniques There are generally two techniques which are used. 1. Direct Transfer: In this technique, a soft to firm catheter, loaded with embryos by the embryologist is directly negotiated through the cervix under ultrasound guidance. No outer sheath is used. The technique may become impractical in difficult embryo transfer with cervical or uterine distortions. The technique is less popular now (Fig. 3).


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Fig. 4  After loading technique

Ultrasound probe

Outer Sheath

Endometrial Cavity

Transferred embryo Inner Catheter

2. After loading technique: In this technique, an outer stiffer coaxial sheath is inserted in the cervix. A very soft inner fine catheter is loaded with embryos. This soft catheter is threaded through the outer sheath into the uterus up to the mid endometrium and embryos are released [3]. In case of very difficult cervices, a stylet may be used to further stiffen the catheter. The most common technique being used routinely is after loading technique (Fig. 4). Now almost all ETs are done under ultrasound guidance which is useful because of the following reasons [4]: 1. A catheter can curl without the clinician realizing the problem and embryos can be deposited at a wrong site or even in cervix. When done under ultrasound guidance, one can ensure proper positioning of catheter, 2. Fluid bubble containing the embryo(s) can be seen at the top of the uterine cavity as the catheter is rotated and then removed giving confidence to the clinician as well as patients.

2 Scheduling and Consents The couple is explained about the need, the steps of procedure (Fig. 5). The counselling and consent include following important aspects: • • • • •

Availability of number and quality of embryos. Pros and cons of different days of embryo (day-2, day-3, or day-5) transfer. Number of embryos to be transferred. Any doubts regarding procedure are to be cleared. Freezing or discarding of supernumery embryos (for which the consent is separately taken).


Embryo Transfer Fig. 5  ET counselling








Fig. 6  Fresh embryo transfer timeline

• For FET, consent is taken for thawing of embryos and quality of embryos to be expected after thawing in case of FET and risk of cancellation if no embryos available. • Chances of implantation and miscarriage after transfer. • Consent is also taken for embryo transfer procedure. In case of fresh embryo transfer, patient is started on Inj. Progesterone (P) 50 mg IM after oocyte retrieval (OR) on the same day which is labelled “Day 0” and scheduling is done on “Day 3” for cleavage stage and “Day 5” for blastocyst transfer (Fig. 6). For a frozen embryo transfer, the endometrium is prepared in a natural, modified natural or hormone replacement cycle with estradiol valerate and once judged to be appropriate, i.e., over 8 mm in thickness, progesterone is started and ET scheduled according to D3 or D5 transfer (Fig. 7). On the day of ET, number of embryos to be transferred and grading of embryos is done as follows:



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Fig. 7  Frozen embryo transfer timeline

2.1 Day of Transfer Embryo transfer may be carried out on day-2, day-3, or day-5. The oocyte collection day is considered as day-0. 1. Day-5 embryo transfer also known as blastocyst transfer is ideal as it helps to select the best growing embryo. It simulates a natural cycle where blastocyst is formed in uterus while embryo on day-2 and day-3 development occurs in fallopian tube. Another advantage is that uterine contractions settle by day-5 after OR and hence may be ideal time. 2. Day-2 or day-3 embryo transfer is done when number of embryo is too low to allow selection up to day-5.

2.2 No. of Embryos to be Transferred The number of embryos may vary from 1 to 3. Ideally, it should be a SET to avoid multiple pregnancies. Various factors to be considered to decide the number of embryo to be transferred are 1. Outcome category depending upon infertility pathology. 2. Patient’s age or oocyte age in case of oocyte donation. 3. BMI. 4. Previous failures. 5. Embryo quality and development stage.

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6. M-II rate. 7. Euploid status if known. 8. Legislation of the country (In India, maximum number allowed is three cleavage stage and two at blastocyst stage).

2.3 Recommendations for the Limit to the Number of Embryos to Transfer As per ASRM guidelines, number of embryos to be transferred can be decided as shown in Table 1: Since embryo biopsy is not routinely done, euploidy status is generally not known. At our centre, following guidelines are considered to decide number of embryos (Table 2). Table 1  ASRM guidelines for number of embryos for ET Type of embryo available and patient characteristic Cleavage stage embryos Euploid Favourable prognosis (Euploidy status not known) All others Blastocyst stage embryos Euploid Favourable prognosis All others

35 >29 All other cases 160° predicts an easy transfer. Difficulty level is directly related to the acuteness of the angle in both ante or retroverted or laterally flexed uteri.

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Fig. 10  Easy transfer. (a) The upper sketch is a diagrammatic representation of position of the ultrasound probe on abdomen and the lower part of the sketch represents the outline of pelvic organs (urinary bladder and transverse section of uterus) as seen on ultrasound image. (b) The transabdominal ultrasound image of a transverse section of uterus as seen posterior to urinary bladder. The uterus is also in the centre and there is no lateral deviation. (c) Diagrammatic representation of utero-cervical angle corresponding to the ultrasound image shown in Fig 10b. (d) Ultrsound image of the utero-cervical angle. A uterus positioned in centre associated with a straight uterocervical angle of >160o predicts an easy transfer

As an example, there is an acute angle of 100° at the level of the internal os. One can anticipate a difficult ET and select a malleable metal catheter sheath, or there may be a requirement of stylet. It is easy to assess anteversion and retroversion and flexion of the uterus. But it is difficult to assess lateral deviation and flexion (Fig. 11). Common reasons for difficult embryo transfers are acute uterocervical angle, presence of cervical stenosis, or anatomical distortion of the cervical canal because of Mullerian abnormalities, nabothian cysts, or pre-existing false passages. Documentation of trial embryo transfer findings is of utmost importance as the actual transfer usually takes place much later which may be few months. A pictorial diagram of projected trajectory in case of a difficult transfer is very helpful (Table 5).




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Fig. 11  Difficult transfer. (a) Diagrammatic representation of ultrasound probe kept in left iliac fossa and position of cervix in relation to bladder, (b) Probe positioning on abdomen, (c) Image if transverse section of uterus produced in case of lateral deviation of the fundus, and (d) Diagrammatic representation of uterocervical angle in a deviated uterine fundus Table 5  Trial ET record Date: Done during: Outer catheter Uterocervical length Uterocervical angel Speculum type and direction Direction Cervix Vaginal discharge

Surgeon: Natural/Progynova cycle Inner catheter

Easy/difficult Healthy/cervicits Normal/abnormal


Embryo Transfer


4 Embryo Transfer Procedure Embryo transfer is the final most important step for the successful IVF cycle and one of the main variables affecting pregnancy rate and implantation during IVF treatment. The requirements of ET procedure are as: 1. Setup of ET OT or ET suit (Fig. 12). 2. Setup of ET in IVF lab. 3. Equipment and disposables. 4. Personnel.

4.1 Setup for ET OT or ET Suit The objective of the setup is to provide transfer of embryos from laboratory to the ART physician’s hands and to the woman’s uterus without undue exposure, accident, or delay. The requirements include: • • • • •


A clean room environment. All aseptic precautions taken in an operation theatre are to be observed. The ET suit needs to be free of embryo toxic volatile organic compounds. The ET suit should be cosy, comfortable, and patient friendly. The ET suit is connected to IVF laboratory through a transfer box which serves as a conduit for safe and quick transfer of loaded catheters from the embryology laboratory.



Fig. 12  ET suite. (a) Lithotomy table. The ET suit requires an adjustable table with a gynae cut to accommodate transvaginal procedures and with leg stirrups for making lithotomy position. (b) Other OT equipment: In addition, the room needs a trolley to set up equipment, waste disposal bins according to bio-medical waste standards, stools for the surgeon and storage for disposables. A set up for general anaesthesia is also present which may be required very rarely for ET. (c) Ultrasound machine-The ET suite should be equipped with an ultrasound machine. Any routine good 2-Day USG machine with a transabdominal convex probe can suffice


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4.2 Setup for ET Embryology Laboratory Same embryology lab as occyte retrieval is used to load embryos in catheter and is passed to physician (Fig. 13). The main equipment required includes a workstation, CO2 incubators, micromanipulator with an inverted microscope or LASER (Figs. 14, 15 and 16). a


Fig. 13  IVF lab for ET. (a, b) The IVF laboratory is fitted with a class 1000 HVAC system (3000 cfm) that is capable of removing 99.97% of particles larger than 0.3 μm in diameter. The HVAC system also ensures passage of clean air through a carbon filter to ensure filtration of VOCs

Fig. 14  Laminar flow workstation

Embryo Transfer





Fig. 15  Inverted microscope and micromanipulator. (a) Narishige micromanipulator with an Olympus inverted microscope. (b) RI-Integra Micromanipulator with LASER. (c) Laser hatching, inverted microscope is fitted with Hoffman modulation and contrast system is useful for studying the embryo morphology and embryo selection before transfer. A direct image transmission to a TV is used to show embryos to the couple and also for training and witnessing. Micromanipulator and LASER system is used for assisted hatching if required



Fig. 16  Incubators. (a) Classical incubator: these are CO2 incubators used for culture, dish making, and media equilibration purposes. (b) Benchtop incubators used for culture in Trigas. The embryos are cultured in Tri-gas bench top incubators. These have a capacity to accommodate eight 30 mm dishes at one point of time. It is always placed strategically so that it is comfortable to open the individual chambers and transfer dishes from the incubator to the workstation without any risk of accident. All individual chambers are labelled properly so as to ensure precise patient identification

A laminar air flow is an enclosed workstation that is used to create a contamination-­ free zone for working. The room air passed through HEPA (High Efficiency Particulates Air) is fed into the working chamber by a unidirectional vertical descending flow (99.97% efficiency for 0.3 μm particles). The workstation is fitted


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with a stereozoom microscope and supplied with a heated table top (37 °C) that aids in embryo loading. The loading of embryos can be viewed through screen so it is a useful tool for witnessing the procedure (Fig. 14).

4.3 Equipment and Disposable 1. ET Catheter (Labotech 320,220, Wallace). 2. Falcon 60 mm dish (cat 3002). 3. 1 mL disposable syringe 4. Transfer medium (Vitrolife, Medicult). 5. Sterile working cabinet with laminar airflow and heating stage. All the media and disposables have to be tested against embryo toxicity and certified IVF grade (Fig. 17).

4.4 ET Catheter Choosing an appropriate catheter is a very important step in the ET procedure. Commercially available embryo transfer catheters can be divided into firm and soft embryo catheters. Most of the catheters now have a very soft inner catheter. There are various other modifications available for difficult ET. Some of these come with inner stylet or air bubbles in the sheath for better visualization. Ideally, the embryo transfer catheter should be selected on the basis of trial ET.

Fig. 17  Embryo transfer disposables. Showing all disposables required for one embryo transfer on workstation: Culture Media, Mineral Oil, Culture Dishes, Droppers, Micropipette, Micro-tips, Stripper, Handling pipettes, ET Catheter, BD Syringes

Embryo Transfer


• Firm embryo transfer catheters: Historically, the ET was done with stiff or firm catheters like Tom-cat catheter. Firm embryo transfer catheters are now used for difficult cases. • Soft embryo transfer catheters: The soft embryo transfer catheters produce better results probably due to reduced trauma to endometrium. They follow the natural curvature of uterine cavity better than the firm embryo transfer catheters. There are soft embryo transfer catheters available which have an echogenic tip and can be seen very clearly on ultrasound without the need to move catheter to identify the tip. Soft catheters are commercially available from various manufacturers. We in our center use Labotect soft catheters (Fig.  18a, b) with malleable outer sheath and markings in centimeters for precise guidance. These soft catheters are available in two different sizes and used according to the expected length of the uterine cavity. Gynetics flexitip embryo transfer metal sheath and soft inner catheter (Fig. 18c, d). In difficult transfers, it is better to use a flexible metal sheath which helps to overcome the subtle but definite resistance encountered by the soft outer sheaths. The embryos are loaded and transferred through a soft inner catheter. Some other ET catheters available are (Figs. 19, 20, 21, 22, 23, 24 and 25): a




Fig. 18  Embryo transfer catheters. (a) Soft embryo transfer catheters in different sizes. (b) Close up of the catheter sheath and inner catheter showing markings on inner and outer catheter. (c) Flexible metal catheter sheath along with inner soft catheter. (d) Close up of the catheter sheath with flexible metal tip and inner soft catheter


Fig. 19  Wallace classic soft embryo transfer catheters

Fig. 20  Wallace sure view ultrasound visible embryo transfer catheters

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Fig. 21  Wallace sure-pro supported embryo transfer catheters Fig. 22  Guardia™ access embryo transfer catheter


80 Fig. 23 Soft-Pass™ embryo transfer catheter set

Fig. 24  Guardia™ access embryo transfer catheter

Fig. 25  Guardia™ access embryo transfer catheter with internal support cannula

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Embryo Transfer

4.5 ET Transfer Procedure 4.5.1 IVF Lab Procedure Steps: 1. The selected embryos are graded and transferred to single droplet of transfer media. 2. Take 1 mL of BD syringe and release if there is any air. 3. Embryo transfer catheter is attached to syringe. 4. Place the embryo containing dish on sterile working cabinet. 5. Bring the embryo transfer catheter close to media and draw some media and then embryo into it with minimal amount of medium. 6. Then the embryologist hands over the catheter to the clinician to carry out the transfer. 7. When the catheter is returned after the procedure, catheter is carefully checked under microscope. 8. The syringe is detached from catheter and medium is drained into petri dish via catheter. Check the medium thoroughly to confirm the transfer of embryos. 9. If the embryos are retained in catheter, they should be reloaded into a new catheter, and the transfer procedure is repeated.

4.6 Loading Techniques Most of IVF laboratories use one of the following loading techniques (Figs. 26, 27 and 28). 1. The syringe is filled with medium right up to the proximal part of embryo transfer catheter. It is followed by 2.5 μL of air followed by 7.5 μL of embryos containing medium followed by 2.5 μL of air and eventually followed by 2.5 μL of culture medium at the tip of catheter (Fig. 26).




Fig. 26  Embryo loading with air columns



U. N. Jindal et al. SYRINGE EMBRYO









Fig. 27  Continuous embryo loading without air columns

Fig. 28  Continuous embryo loading without air columns and with empty syringe

2. The syringe and entire embryo transfer catheter is filled with air followed by 7.5 μL of embryo containing culture medium followed by 1.5 μL of air at the tip (Fig. 28). The use of air brackets around the embryos containing medium has been debated to be beneficial for the success of embryo transfer, • by protecting the embryo from the cervical mucus, • from accidental discharge before entering the endometrial cavity. On the other hand, the supporter of fluid only method for loading believe that the introduction of even a small amount of air in uterus could be a non-physiological factor with a deleterious effect on embryo implantation. The presence of air in the transfer catheter could increase the likelihood of retained embryos, reactive oxygen, and movement of embryos to other areas away from uterus. Currently, however, embryo transfer implies a reduction of total transfer volume, including both transfer of media and air. In our laboratory, the total volume of

Embryo Transfer


transfer is small and includes only 6–7 μL of medium containing the embryos and 2.5–5.0 μL of air that almost unavoidably exists within catheter when a full column of medium is not used.

4.7 Embryo Transfer Clinical Procedure After selection of embryos and all paper work, the stage is set for actual procedure. Patient with partly full bladder is taken to the ET suit and made to lie down in the lithotomy position. The nurse checks the adequacy of fullness of bladder and gives a go ahead to the ART specialist and embryology laboratory. The instrument trolley is set. The physician and sonologist take their places. A re-evaluation of the difficulty and trajectory of ET catheter is made and catheter is selected. Various steps of procedure are shown in pictures. The trolley is covered with a sterile sheet and following equipment is kept: appropriate- sized Cusco’s speculum, Sponge holder, bowl with normal saline for cleaning along with sterile gauze (Fig. 29). A sterile sheet to be put under patient, The ET catheter is opened only after the final order by the physician. The various steps in ET OT are as: (a) The ART physician sits on the vaginal end of the patient put to lithotomy position and inserts the vaginal speculum and cleans the cervix and vagina with sterile gauze using saline. Removal of mucus from the endocervical canal is optional.

Fig. 29  Embryo transfer trolley


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(b) Nursing assistant opens the packaging and gives the inner catheter to the embryologist via pass box and outer sheath is given to the ET physician with all sterile precautions. Appropriate-sized Cusco’s speculum is inserted and locked. The sonologist keeps the view of cervical canal and endometrium in same straight line (Fig. 30). (c) The external catheter sheath is inserted and positioned at the level of internal os under transabdominal ultrasound guidance (Fig. 31). Once the ART Physician is satisfied that the external catheter is appropriately placed, message is given to the laboratory to load the internal ET catheter with the selected embryos (Fig. 32). Loaded embryo transfer catheter is handed over to the specially assigned nurse in the ET suite via a pass box (Fig. 32). Utmost care is again taken while transferring the loaded catheter to the ET surgeon. Fig. 30  Uterus in sagittal plane

Fig. 31  Inserting catheter external sheath

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Fig. 32  Loaded catheter being handed over to ET suite





Fig. 33  Loaded catheter being threaded in external catheter. (a) Transabdominal image of ultrasound showing outer catheter sheath in cervix and endometrium in line of catheter. (b) Inner catheter being threaded in the outer catheter sheath. (c) Inner catheter threaded in the outer catheter sheath. (d) Advancement of inner catheter under ultrasound guidance


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Fig. 34  Embryo transfer ultrasonic view

Once the inner catheter is transferred to the ET surgeon, it threaded through the already placed outer sheath in a smooth way (Fig. 33a). Ultrasound focuses the uterus with external sheath in situ (Fig. 33b, c). After threading of internal catheter with embryos through the sheath (Fig. 33d), advancement of internal catheter in the endometrium under ultrasound guidance is done. This is the most important step during which the coordination between clinician and sonologist is very crucial. Once internal catheter placement is satisfactory, the embryos are ejected from the internal catheter with a gentle push of plunger. The ejection of embryos along with air and the small media droplet can be very easily seen with ultrasound (Fig. 34). Cautions: • The syringes to be used must be squeezed in a controlled fashion so as not to ‘pop’ the embryos with such force that they are damaged or thrust into fallopian tubes. • The time interval between loading and discharging the embryo should not exceed 120  s. It has been shown to be detrimental on embryos as it may result in a change in the temperature, pH, and osmolarity of the media in which the embryos are loaded. • The osmolarity of medium must be within limits of 275–305 mOsm with a total variation of not more than 30 mOsm. A lowering in osmolarity during the procedure may cause an increase in the size of embryo relative to the volume of cytoplasm, and as a result, cytoplasmic blebs may be formed in order to reach adequate nucleocytoplasmic (N/C) ratio for entering mitosis. However in doing so, the embryo loses not only the cytoplasm but also mRNA and proteins which are necessary for future development. pH must be within 7.2–7.5 with a maximal variation of 0.4. Reduction in temperature can cause irreversible disruption of the mitotic spindle with possible chromosomal dispersal which may contribute to a high rate of preclinical and spontaneous abortions.

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• A large volume of transfer media and a large air interface may result in expulsion of embryos into cervix. • Note and record the presence of blood or mucus on embryo transfer catheter. The presence of blood on the outside of the embryo transfer catheter tip may indicate a difficult embryo transfer. The blood or mucus on the embryo transfer catheter tip is associated with a higher incidence of retained embryo as embryo can be buried in mucus.

4.8 Remaining Embryos The embryos of grade 1 and 2 which remains after the embryo transfer procedure may be cryopreserved. The remaining cleaved embryos unsuitable for freezing can be kept in culture and scored till day-6, and if they develop into blastocysts by day-6, it can be preserved. A double identity check of the patient, the patient file, and the culture dish(es) is mandatory immediately before the transfer [8, 9].

References 1. Ghanem ME, Ragab AE, Alboghdady LA, Helal AS, Bedairy MH, Bahlol IA, Abdelaziz A.  Difficult embryo transfer (ET) components and cycle outcome. Which is more harmful? Middle East Fertil Soc J. 2016;21(2):114–9. 2. Practice Committee of the American Society for Reproductive Medicine. ASRM standard embryo transfer protocol template: a committee opinion. Fertil Steril. 2017;107:897–900. 3. Toth TL, Lee MS, Bendikson KA. ASRM survey of current society for assisted reproductive technology practices. Fertil Steril. 2017;107:0015–282. 4. Strickler RC, Cat Christianson RN, Crane JP, Curato A, Knight AB, Yang V. Ultrasound guidance for human embryo transfer. Fertil Steril. 1985;43(1):54–61. 5. Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2001;26(6):1270–83. 6. Practice Committee of the American Society for Reproductive Medicine. Performing the embryo transfer: a guideline. Fertil Steril. 2017;107:882–96. 7. Toth TL, Malinda MD, Lee S, Bendikson KA, Richard H, Reindollar for the American Society for Reproductive Medicine Embryo Transfer Advisory Panel. Embryo transfer techniques: an American Society for Reproductive Medicine survey of current Society for Assisted Reproductive Technology practices. Fertil Steril. 2017;107:1003–11. 8. Cristina Magli M, Van den Abbeel E, Lundin K, Royere D, Van der Elst J, Gianaroli L. Committee of the Special Interest Group on embryology revised guidelines for good practice in IVF laboratories. Hum Reprod. 2008;23(6):1253–62. 9. Kava A, Braverman FM, Rodríguez I, Alvarez M, Barri PN, Coroleu B.  What is a difficult transfer? Analysis of 7,714 embryo transfers: the impact of maneuvers during embryo transfers on pregnancy rate and a proposal of objective assessment. Fertil Steril. 2017;107(3):0015.

Intrauterine Insemination Surveen Ghumman and Parul Aggarwal

Intrauterine insemination refers to the process of placing high quality, motile, concentrated sperm, free of seminal plasma, and debris into the uterine cavity closer to ovulated oocytes. This procedure increases the available spermatozoa at the site of fertilization by as much as 25%. The clinical indications for intrauterine insemination are listed in Table 1. Table 1  Indications of IUI

Female factor  • Anatomic defects of vagina or cervix  • Antisperm anibodies in cervix  • Sexual dysfunction  • Mild to moderate endometriosis  • Endocrine anomalies  • Ovulatory dysfunction Male factor  • Male subfertility: e.g. mild oligozoospermia, asthenozoospermia, or teratozoospermia  • Anatomic defect of penis, e.g. hypospadias  • Sexual/ejaculatory dysfunction  • Retrograde ejaculation  • Low semen volume  • Immunological factors  • Azoospermia—for donor insemination

S. Ghumman (*) IVF and Reproductive Medicine, MAX Superspeciality Hospitals, New Delhi, India P. Aggarwal Medicover IVF Centre, Delhi, India © Springer Nature Singapore Pte Ltd. 2023 S. Ghumman (ed.), Atlas of Assisted Reproductive Technologies,


90 Table 1 (continued)

S. Ghumman and P. Aggarwal Other factors  • Unexplained infertility  • Combined subfertility factors  • Serodiscordant couples with hepatitis B and HIV

1 Follicle Monitoring and Timing of IUI The IUI process commences with an ultrasound scan on day-2 of menses to look for ovarian status and to rule out ovarian cysts. Ovulation induction with oral ovulogens like clomiphene citrate or letrozole or injectable gonadotropin is administered from day-2 or day-3 of the cycle. Success rates increase with ovulation induction [1, 2]. Ultrasound scan for follicular monitoring is done, in order to track follicle development and endometrial thickness. Once the follicle size and endometrial thickness reach threshold sizes of 18–20 mm and 8 mm, respectively, 5000 IU of injection hCG is administered to induce final maturation of oocyte and follicular rupture which is expected to occur around 36 h. After ultrasound confirmation of follicle rupture, intrauterine insemination is performed between 36 and 40 h. Consent is taken before the procedure and complications are explained (Table 2).


Intrauterine Insemination Table 2  Complications of IUI

IUI complications  • Uterine cramping −5%  • Spotting −1%  • Gastrointestinal upset- 0.5%  • Infection 0.2%  • Multiple gestation  • Ectopic gestation

2 Sample Collection For sample collection from husband, a designated semen collection room is necessary with the sample container marked with an identification label. Patient should be instructed to legibly write the names of both husband and wife on the envelope and on the container sticker (Fig. 1). During sample collection, in case of mishandling such as sample spillage on the ground, patient must immediately inform the technician. Importantly, if patient prefers to use a condom, it should be ensured that he uses a non-toxic one without spermicidal jelly.


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Fig. 1  Non toxic container with collected semen sample

3 Semen Analysis After sample collection, semen analysis parameters, viz., sperm count, motility, and morphology should be assessed to determine the type of processing required (Fig. 2). In order to prevent gamete mixing, it is important to ensure that the sample of one patient is collected and processed at a time. Proper labelling of all disposables such as pipettes and test tubes must be done carefully. Double witnessing is essential.

Intrauterine Insemination


Fig. 2 Microscopic examination of semen

4 Semen Processing The semen sample is processed using sperm preparation techniques such as double density gradient centrifugation or swim-up technique by centrifugation (Figs. 3 and 4).

94 Fig. 3  Centrifugation of semen sample

Fig. 4  Centrifuged semen sample with pellet

S. Ghumman and P. Aggarwal

Intrauterine Insemination


5 Procedure The instruments are set on a sterile trolley (Fig. 5). Patient should have a partially full bladder. She is positioned in a semi lithotomy position in the insemination room (Fig. 6). Perineum is cleaned with saline. No povoiodine is used as it is harmful to the sperm. Cuscos speculum is use for visualizing the cervix which is cleaned with normal saline. The prepared sample is 0.3–0.5 mL which is then loaded after checking the identification of the sample (Fig. 7). After cleaning the cervix with a swab, it is irrigated with buffer. The catheter is flushed with media and then loaded. The fully assembled catheter is passed gently through the internal os followed by slow injection of the loaded sample over a 1–2 min period (Fig. 8). The catheter is then slowly removed after 30 s and patient is advised bed rest for 10–15 min. It is important to note that there is no need of prolonged bed rest, head low, pain-killers, antispasmodics, or antibiotics. Fig. 5 Instruments required for IUI

Fig. 6 Semi-lithotomy position for IUI

96 Fig. 7  Loading of semen sample in IUI catheter

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Intrauterine Insemination


Fig. 8  Insertion of IUI catheter in cervix

6 Post Procedure Instructions After the procedure is complete, the patient is given a detailed report of the processed semen. Post IUI instructions include luteal phase support and next follow-up date is discussed. They are advised a pregnancy test on the 16th day post IUI.


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7 Types of IUI Catheters IUI catheters can be soft or firm. They come with stylets to help negotiate the catheter through the cervical canal (Figs. 9 and 10). Soft catheters are used for easy IUI [3]. Firm ones are needed to negotiate a curved cervix. Embryo transfer catheters may be used where the canal is curved and requires negotiation (Fig. 11). In very difficult IUI, where there may be a cervical stenosis, metal catheter can also be used [4]. Fig. 9  IUI catheter with stylets

Fig. 10  Stylets used for IUI catheter

Intrauterine Insemination


Fig. 11  Embryo transfer catheter which can be used for IUI purpose

8 Strategies to Overcome Difficulties in IUI • Fill up bladder in acutely anteverted uterus to straighten uterus. • Use ultrasound guidance to see curvature of the cervical canal and the position of the catheter. • Stylet may be used with catheter. • Change IUI catheter to a firm one such as Maklers or metal or switch to an embryo transfer catheter which can negotiate better. • Use the tenaculum and give traction to manipulate the utero-cervical angle and also stabilize the cervix. Use of tenaculum should be avoided as far as possible as it may induce uterine contractions. • Consider cervical dilatation with smallest dilator or sound which helps to identify the direction and also to negotiate the stenosis. However, this should be avoided as it can cause trauma. • Make a note on the paper and consider for cervical dilatation on the first or second day of next menses.

9 Double Vs. Single IUI A second IUI may be considered after 24 h in some cases if follicle has not ruptured. Studies have shown that double IUI may increase the pregnancy rate significantly in patients with male factor infertility [5]. However, single IUI acts as efficiently as double IUI in patients with idiopathic infertility. IUI is the basic procedure used in infertility to overcome a wrong timing, difficulty in intercourse, aberrant ovulation, or a semen factor. It has a success rate of about 15% in unexplained infertility and mild male factor. Success rates may drop with pelvic pathologies such as tubal factor or endometriosis. It is important to counsel couples about the success rates before taking them up for the procedure.


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References 1. Ayeleke RO, Asseler JD, Cohlen BJ, Veltman-Verhulst SM. Intra-uterine insemination for unexplained subfertility. Cochrane Database Syst Rev. 2020;3(3):CD001838. PMID: 32124980. 2. Soysal C, Ozmen U.  Intrauterine insemination in ovulatory infertile patients. Niger J Clin Pract. 2018;21(10):1374–9. 3. Abou-Setta AM, et al. Intrauterine insemination catheters for assisted reproduction: a systematic review and meta-analysis. Hum Reprod. 2006;21:1961–7. 4. Van der Poel N, Farquhar C, Abou-Setta AM, Benschop L, Heineman MJ. Soft versus firm catheters for intrauterine insemination. Cochrane Database Syst Rev. 2010;11:CD006225. PMID: 21069687. 5. Zavos A, Daponte A, Garas A, et  al. Double versus single homologous intrauterine insemination for male factor infertility: a systematic review and meta-analysis. Asian J Androl. 2013;15(4):533–8.

Sperm Retrieval Techniques Fabio Coltro Neto, Bárbara Ferrarezi, and Sandro C. Esteves

1 Introduction The development of intracytoplasmic sperm injection (ICSI) was a breakthrough for men with severe male factor infertility [1]. Equally important was the application of ICSI to azoospermic men and the confirmation that spermatozoa taken from either the epididymis or testis were capable of normal fertilization and pregnancy [2, 3]. Azoospermia is clinically divided into obstructive and non-obstructive. Obstructive azoospermia (OA) is characterized by normal spermatogenesis in which either a mechanical blockage exists somewhere between the rete testis and ejaculatory duct or the epididymis/vas deferens are absent (total or partial agenesis). Acquired OA is due to vasectomy, failure of vasectomy reversal, post-infectious diseases, surgical procedures (scrotal, inguinal, pelvic, and abdominal regions), and trauma. Congenital OA is due to congenital absence of the vas deferens (CAVD), ejaculatory duct or prostatic cysts, and Young’s syndrome [4]. Non-obstructive azoospermia (NOA) comprises a spectrum of severe testicular disorders resulting from various causes, including endocrine and genetic abnormalities, post-infectious, trauma, environmental toxins, gonadotoxin exposure (radiation, chemotherapy), cryptorchidism, varicocele, and idiopathic [4–6].

F. C. Neto · B. Ferrarezi Division of Urology, Department of Surgery, University of Campinas (UNICAMP), Campinas, Brazil S. C. Esteves (*) Division of Urology, Department of Surgery, University of Campinas (UNICAMP), Campinas, Brazil ANDROFERT, Andrology and Human Reproduction Clinic, Referral Center for Male Infertility, Campinas, SP, Brazil e-mail: [email protected] © Springer Nature Singapore Pte Ltd. 2023 S. Ghumman (ed.), Atlas of Assisted Reproductive Technologies,



F. C. Neto et al.

Several methods have been developed to retrieve epididymal and testicular sperm from azoospermic men. Among them, percutaneous epididymal sperm aspiration (PESA) [7] and microsurgical epididymal sperm aspiration (MESA) [2] are used to retrieve sperm from the epididymis. In contrast, testicular sperm aspiration (TESA) and testicular sperm extraction (TESE) are the methods to harvest sperm from the testicle in OA [7, 8]. Additionally, open testicular sperm extraction (TESE) using single or multiple biopsies [9–11] and TESE with microsurgery (microdissection testicular sperm extraction; micro-TESE) are methods to harvest sperm from men with NOA [12–14]. This chapter describes the commonly used sperm retrieval methods: PESA, MESA, TESA, TESE, and micro-TESE.

2 Sperm Retrieval Techniques 2.1 Materials, Equipment, and Reagents 2.1.1 Operating Room • • • • • • • •

Sterile surgical gloves and syringes (1 mL, 20 mL). 0.7 × 25 mm, 0.45 × 13 mm, 1.2 × 40 mm (TESA/PESA) disposable needles 2% Lidocaine Heating block for test tubes. Syringe holder (TESA, see Fig. 3). Surgical loupe (PESA and TESA). Operating microscope (micro-TESE and MESA). Microsurgery instruments (micro-TESE and MESA).

2.1.2 IVF Laboratory • • • • • • • • • • • •

Petri dishes. Disposable serological pipettes. Pipettor 1–200 μL and sterile tips. Pipetting device. 6-mL sterile centrifuge polystyrene tubes with caps 0.7 × 25 mm needles and tuberculin syringes Marker pen. ICSI micropipettes. Laminar flow cabinet. Warming plate. Stereomicroscope. Centrifuge.

Sperm Retrieval Techniques


• Inverted microscope equipped with Hoffman modulation contrast and electro-­ hydraulic micromanipulators. • HEPES-buffered Human Tubal Fluid (HTF) and Human Serum Albumin (HSA). • Mineral oil. • Polyvinylpyrrolidone (PVP) solution. • Colloidal density gradient. • Erythrocyte lysing buffer. • Collagenase. • Pentoxifylline solution. 2.1.3 Laboratory Setup Note: Sterile handling conditions under a laminar flow cabinet or cleanroom environment should be used during all laboratory steps. • A 10-mL (for PESA) or 20-mL (TESA/TESE/MESA) HEPES-buffered protein-­ supplemented (5% HSA) sperm culture medium is prepared and kept at 37 °C. • A 5-mL aliquot of sperm culture medium is transferred to a 6-mL polystyrene tube and sent to the operating room (buffered sperm media is used for flushing the syringe + needle before aspiration and incubating epididymal aspirates or testicular specimens). • Two Petri dishes are placed on a warm surface (37 °C) inside the workstation (for PESA only). • 4-well dishes (e.g., Nunc) are prepared by adding 0.5 mL sperm medium-­aliquots to each well (TESE). Keep them on a warm surface (37 °C) inside the workstation. • Two tuberculin syringes connected with a 13-gauge needle are prepared to be used as tools for mincing and squeezing seminiferous tubules in TESA/TESE processing.

2.2 PESA Note: We perform PESA under local and intravenous anesthesia [15, 16]. • A 10-mL solution of 2% lidocaine is injected around the spermatic cord near the external inguinal ring upon patient unconsciousness. The epididymis is stabilized between the index finger, thumb, and forefinger while the testis is held with the palm of the hand. • A 13-gauge needle attached to a 1-mL tuberculin syringe is inserted into the epididymis through the scrotal skin. Loupe magnification is used to avoid injuring small vessels seen through the skin (Fig. 1). • Negative pressure is created, and the tip of the needle is gently moved in and out within the epididymis until fluid enters the syringe. The amount of epididymal


F. C. Neto et al.

Fig. 1  Percutaneous epididymal sperm aspiration (PESA). The flow chart illustrates PESA surgical steps. (Reprinted with permission, ANDROFERT© 2020. All rights reserved)

fluid obtained during aspiration is often minimal (~0.1 mL), except in cases of CAVD where more fluid can be aspirated. • The needle is withdrawn from the epididymis, and the aspirate is flushed into a 0.5 mL 37° C sperm medium. • The tube containing the epididymal aspirate is transferred to the IVF lab. PESA is repeated at a different site of the same epididymis (from cauda to caput) or the contralateral one until an adequate number of motile sperm is retrieved. If PESA fails to retrieve motile sperm for ICSI, TESA is performed at the same operative time. A short movie depicting the main steps of the procedure can be found at http:// [15].

Sperm Retrieval Techniques


2.3 MESA Notes: We perform MESA under local and intravenous anesthesia [15, 16]. An operating microscope and microsurgery technique are used throughout the procedure, as previously described [15, 16] (Fig. 2). • A 10-mL solution of 2% lidocaine is injected around the spermatic cord near the external inguinal ring upon patient unconsciousness. After anesthetic blockade of the spermatic cord, the anterior scrotal skin is stretched, and the skin and tunica vaginalis are infiltrated with 2  mL of 2% lidocaine. A transverse 3-cm incision is made through the anesthetized layers, and the testis is exteriorized. • The epididymal tunica is incised, and an enlarged tubule is selected. An enlarged  epididymal tubule is dissected and opened with sharp microsurgical

Fig. 2  Microsurgical epididymal sperm aspiration (MESA). After the testis and epididymis are exposed, a dilated epididymal tubule is dissected and opened. The fluid is aspirated, diluted with sperm medium, and sent to the laboratory for examination. (Reprinted with permission, ANDROFERT© 2020. All rights reserved)


F. C. Neto et al.

scissors. The fluid that flows out of the tubule is aspirated with the aid of a silicone tube or a needle attached to a tuberculin syringe. • The aspirate is flushed into a tube containing warm sperm medium and transferred to the laboratory for examination. MESA can be repeated at a different site on the same epididymis (from the cauda to caput regions) or the contralateral epididymis until adequate number of motile sperm is retrieved. If MESA fails to retrieve motile sperm, TESA or TESE can be performed as part of the same procedure. However, MESA often provides enough sperm for cryopreservation. A single MESA usually enables the retrieval of many high-quality sperm that can be used for ICSI or intentionally cryopreserved for subsequent ICSI cycles.

2.4 TESA Note: We perform TESA under local and intravenous anesthesia [15], as described elsewhere. • After anesthetic blockade of the spermatic cord, the testis is stabilized between the index finger, thumb, and forefinger while the anterior scrotal skin is stretched. • A 23-gauge needle attached to a 20-mL syringe is connected to a syringe holder. It is inserted through the stretched scrotal skin into the anteromedial or anterolateral portion of the superior testicular pole at an oblique angle towards the medium and lower poles (Fig.  3). Loupe magnification is used to avoid injuring  small vessels seen through the skin. • Negative pressure is created by pulling the syringe holder, whereas the needle is moved in and out within the testis in an oblique plane to disrupt the seminiferous tubules and sample different areas. When a small amount of testicular tissue is aspirated, the needle is gently withdrawn from the testis while the negative pressure is maintained. A pair of microsurgery forceps is used to grab the s­ eminiferous tubules that exteriorize from the scrotal skin, thus aiding in removing the specimen. • The specimen is flushed into a tube containing 0.5–1.0 mL warm sperm medium and is transferred to the IVF lab. TESA can be performed at the contralateral testis at the same operative time if insufficient numbers of spermatozoa are retrieved for ICSI. A short movie depicting the main steps of the procedure can be found at https://

Sperm Retrieval Techniques


Fig. 3  Testicular sperm aspiration (TESA). The flow chart illustrates TESA surgical steps. Percutaneous aspiration of the testicle is carried out by inserting a 40 × 12 mm needle connected to a 20 mL syringe mounted on a syringe holder (e.g., Cameco syringe holder). The testis is firmly held, and the needle is moved in and out in various directions under negative pressure to disrupt and facilitate the extraction of seminiferous tubules. Specimens are sent to the laboratory for mechanical mincing and examination under the inverted microscope for sperm search. (Reprinted with permission, ANDROFERT© 2020. All rights reserved)

2.5 Testicular Sperm Extraction (TESE) Note: We perform TESE under local and intravenous anesthesia [15], as described elsewhere. • The skin and subjacent layers are incised transversally to expose the tunica albuginea, which is opened with the knife. • A small transversal albuginea opening (0.5–1.0 cm incision) is made at the mid-­ testicular pole, and a small sample of the parenchyma is cut off with scissors. • The tunica is closed with a non-absorbable 5–0 running suture (Fig. 4). • Tunica vaginalis, dartos, and skin are sutured with absorbable suture [17, 18].


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Fig. 4  Testicular sperm extraction (TESE). The flow chart illustrates TESA surgical steps. A single or multiple incision(s) is/are made on the tunica albuginea, and one or several testicular biopsies are taken. Specimens are sent to the laboratory for mechanical mincing and examination under the inverted microscope for sperm search. (Reprinted with permission, ANDROFERT© 2020. All rights reserved)

2.6 Micro-TESE Note: We perform micro-TESE under local and intravenous anesthesia, as described elsewhere [14]. For micro-TESE, an operating microscope and microsurgery technique are used throughout the procedure, as previously described [19] (Fig. 5). • After anesthetic blockade of the spermatic cord, the anterior scrotal skin is stretched, and the skin and tunica vaginalis are infiltrated with 2 mL of 2% lidocaine. A transverse 2-cm incision is made through the anesthetized layers, and the testis is exteriorized.

Sperm Retrieval Techniques


Fig. 5  Microdissection testicular sperm extraction (micro-TESE). The flowchart illustrates the micro-TESE surgical steps. Its rationale is to identify focal areas of sperm production within the testes, based on the size and appearance of the seminiferous tubules, with the aid of the operating microscope. A large incision is made in an avascular area of the tunica albuginea, and the testicular parenchyma is widely exposed. The parenchyma is then dissected at ×16 – ×25 magnification to enable the search and isolation of seminiferous tubules exhibiting larger diameters than nonenlarged or collapsed counterparts. These enlarged tubules are more likely to contain germ cells and eventually normal sperm production. Microsurgical-guided biopsies are performed by carefully removing such tubules and sent to the laboratory for examination. The minimal tissue extracted facilitates laboratory processing and sperm search, thus increasing the process efficiency. The initial laboratory step involves mechanical mincing the seminiferous tubules and examining specimens for sperm identification. Optical magnification also reduces the chances of vascular injury by properly identifying testicular blood supply, thus reducing the chances of hematoma formation and testicular devascularization. (Reprinted with permission, ANDROFERT© 2020. All rights reserved)

• A single, large, mid-portion incision is made in an avascular area of the tunica albuginea under 6-8x magnification, and the testicular parenchyma is widely exposed.


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• Dissection of the testicular parenchyma is carried out at 16–25× magnification, searching for enlarged seminiferous tubules, which are more likely to contain germ cells (Figs. 6 and 7). If necessary, the superficial and deep testicular regions may be examined, and microsurgical-guided testicular biopsies are performed by removing the enlarged tubules. If enlarged tubules are not seen, any tubule ­different from the remaining ones in size is excised [11]. If all tubules are identical in appearance, random micro-biopsies (at least three at each testicular pole) are performed. • Each excised testicular tissue specimen is placed in a Petri dish containing sperm media. Specimens are washed grossly to remove blood clots and are sent to the IVF laboratory for processing. A short movie depicting the main steps of the procedure can be found at http:// [19].





Fig. 6  Pre-processing microscopic appearance of seminiferous tubules (Magnification: 100×, inverted optical microscope, Nikon Eclipse Diaphot 300 with phase contrast (Hoffman). (a and b) Thin seminiferous tubules, as usually observed in Sertoli-cell cases only; (c and d) seminiferous tubules of larger diameter than the previous ones (A-B), compatible with the presence of germ cells and mature sperm. (Reprinted with permission from Springer Nature Switzerland AG 2019, Verza Jr. & Esteves, PESA/MESA/TESA/TESE Sperm Processing. In: Z. P. Nagy et al. (eds.), In Vitro Fertilization,­3-­319-­43011-­9_26)

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Fig. 7  Photomicrographs illustrate the association between testicular histology and cell pattern observed after testicular parenchyma processing. In the upper part, the three main histological patterns found in cases of nonobstructive azoospermia are shown: (a) Sertoli cell-only (SCO); (b) Maturation arrest (MA); (c) hypospermatogenesis (HYPO). At the bottom, the corresponding cell pattern usually found in each of these conditions, as seen under the inverted microscopy. In SCO  (d), only Sertoli cells (a), lymphocytes (b), and erythrocytes (c) are usually observed. In MA (e), the presence of a large number of germ cells is seen, and cell differentiation usually stops before sperm formation: (d) spermatogonia / primary spermatocytes, (c) secondary spermatocytes, (f) round spermatids (note the soft contour and prominent acrosome vesicle in the apical portion). It is essential to highlight that isolated foci of active spermatogenesis with differentiation until spermatozoa may be found in SCO and MA. In HYPO (f), the presence of germ cells is reduced. Still, all spermatogenesis stages are present, including spermatozoa: (a) Sertoli cells, (e) secondary spermatocyte, (f) round spermatid, and spermatozoon (highlighted in the circle). Magnification: 400x, inverted optical microscope, Nikon Eclipse Diaphot 300 with phase contrast (Hoffman), and hematoxylin/eosin staining for histological slides at 200 × magnification. (Reprinted with permission from Springer Nature Switzerland AG 2019, Verza Jr. & Esteves, PESA/MESA/TESA/TESE Sperm Processing. In: Z.  P. Nagy et  al. (eds.), In Vitro Fertilization, https://doi. org/10.1007/978-­3-­319-­43011-­9_26)

3 Surgical Complications After SR, postoperative complications include persistent pain, swelling, infection, hydrocele, and hematoma [20–22]. Ultrasound scans performed 3 months after single- or multiple biopsy TESE reveal the presence of intratesticular hematoma in approximately 80% of patients, which tend to resolve spontaneously without compromising testicular function [22, 23]. However, if large-volume parenchyma is extracted, temporary or definitive testicular damage (such as complete devascularization) might decrease serum T levels [20, 24]. TESA and micro-TESE minimize the risk of complications and long-term adverse consequences, including hypogonadism [12, 20, 22, 24–26].


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In micro-TESE, subalbuginea vessels are spared during the testicular opening [16]. The use of optical magnification and microsurgical technique allows preservation of intratesticular blood supply and increases the chances of identifying sperm-­ producing tubules [19, 20, 25]. Hence, SR efficacy is optimized since the risk of complications, and the quantity of tissue removed are reduced. Compared to conventional TESE, the smaller amount of tissue extracted speeds up tissue processing and sperm search [19, 27]. In a large cohort study involving 435 NOA patients subjected to micro-TESE or conventional TESE, postoperative ultrasound examination confirmed that micro-TESE caused fewer acute and chronic testicular changes than TESE [20]. The authors of the study mentioned above reported that although there was an initial reduction in testosterone (T) levels after micro-TESE, such levels return to 95% of their preoperative values in an 18-month follow-up period. These findings have been corroborated by others [28]. Nevertheless, men with severely hypotrophic testes and low serum T levels (e.g., Klinefelter Syndrome) might have a more significant reduction in T levels, thus being at a higher risk of requiring permanent T replacement therapy [29]. In one report involving KS men, serum T levels significantly declined by 30–35% (p 14 mm: Oocytes retrived: Oocyte quality: Decision Sperm quality Injected Fert Cryopreserved Transfer Outcome

Male partner 35 Normozoospermia

1st attempt 1st cycle Antagonist (recagon, cetrotide) 1022 IU 36 h hcG 10 1020 pmol/L 8 6 (Fig. 35a, b) All with intended zona , no PVS and no resistance to injection ICSI Normal 6 MI 4 (Fig. 35c) D3 2 emb; 1X4II, 1X6II (Fig. 35d, e) D3 thaw D5 transfer; D5 1x blast (2BC), 1x cpg (Fig. 35f) Single, MA @ 6 weeks (no FH)


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Fig. 35 (a, b) Oocytes with intended zona, no PVS and no resistance to injection, (c) 2 pronuclei seen following ICSI with oocyte with intended zona, (d) day 3: embryo—4 cell grade 2 (intended zona + crowding of blastomeres), (e) day 3: embryo—6 cell grade 2 (intended zona + crowding of blastomeres), (f) day 5: blast (2BC), compacting

Oocyte Morphology




Fig. 35 (continued)



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Case 6

Age (years) Diagnosis

Female partner 42 NORMORESPONDER

Cycle details: Protocol: Total gonadotropin: Trigger: Days of stimulation: Oestradiol at time of HCG: No. of follicles >14 mm: Oocytes retrived: Oocyte quality: Decision

Fig. 36 (a) Oocytes with dense central granularity, (b) day 3: embryo 2 × 9 cell grade 2, 1 × 8 cell grade 1

Male partner 42 Normozoospermia

8th attempt CCC (clome, Gonal F, menopur) 2100 IU 35 1/2 h HcG 9 1220 pmol/L 6 6 All with dense central granularity (Fig. 36a) ICSI




Oocyte Morphology Sperm quality Injected Cleaved Cryopreserved Transfer Outcome

Normal 6 6 D3 6 embryos D3 thaw D3 transfer; D3 2x9II, 1x8II (Fig. 36b) Beta positive, single, live healthy baby (no postnatal complications)

References 1. Ten J, Mendiola J, Vioque J, de Juan J, Bernabeu R. Donor oocyte dysmorphisms and their influence on fertilization and embryo quality. Reprod Biomed Online. 2007;14:40–8. 2. Loutradis D, Drakakis P, Kallianidis K, Milingos S, Dendrinos S, Michalas S. Oocyte morphology correlates with embryo quality and pregnancy rate after intracytoplasmic sperm injection. Fertil Steril. 1999;72:240–4. 3. De Sutter P, Dozortsev D, Qian C, Dhont M.  Oocyte morphology does not correlate with fertilization rate and embryo quality after intracytoplasmic sperm injection. Hum Reprod. 1996;11:595–7. 4. Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan R. Oocyte morphology does not affect fertilization rate, embryo quality and implantation rate after intracytoplasmic sperm injection. Hum Reprod. 1998;13:3431–3. 5. Balaban B, Ata B, Isiklar A, Yakin K, Urman B. Severe cytoplasmic abnormalities of the oocyte decrease cryosurvival and subsequent embryonic development of cryopreserved embryos. Hum Reprod. 2008;23:1778–85. 6. Esfandiari N, Burjaq H, Gotlieb L, Casper RF. Brown oocytes: implications for assisted reproductive technology. Fertil Steril. 2006;86:1522–5. 7. Rienzi L, Ubaldi F, Martinez F, Iacobelli M, Minasi MG, Ferrero S, Tesarik J, Greco E. Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI. Hum Reprod. 2003;18:1289–93. 8. Esfandiari N, Burjaq H, Gotlieb L, Casper RF. Brown oocytes: implications for assisted reproductive technology. Fertil Steril. 2006;86(5):1522–5. 9. Balaban B, Urman B. Effect of oocyte morphology on embryo development and implantation. Reprod Biomed Online. 2006;12(5):608–15. 10. Otsuki J, Okada A, Morimoto K. The relationship between pregnancy outcome and smooth endoplasmic reticulum clusters in MII human oocytes. Hum Reprod. 2004;19:1591–7. 11. Serhal PF, Ranieri DM, Kinis A, Marchant S, Davies M, Khadum IM. Oocyte morphology predicts outcome of intracytoplasmic sperm injection. Hum Reprod. 1997;12:1267–70. 12. Braga DP, Setti AS, Figueira Rde C, Machado RB, Iaconelli A Jr, Borges E Jr. Influence of oocyte dysmorphisms onblastocyst formation and quality. Fertil Steril. 2013;100(3):748–54. 13. Xu J, Yang L, Zhi-Heng M-NY, Chen J, Sun L. Oocytes with smooth endoplasmic reticulum aggregates are not associated with impaired reproductive outcomes: matched retrospective cohort study. Front Endocrinol (Lausanne). 2021;12:688967. 14. Otsuki J, Nagai Y, Chiba K. Lipofuscin bodies in human oocytes as an indicator of oocyte quality. J Assist Reprod Genet. 2007;24:263–70. 15. Xia P.  Intracytoplasmic sperm injection: correlation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryo quality. Hum Reprod. 1997;12(8):1750–5.


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16. Tartia AP, Rudraraju N, Richards T, Hammer MA, Talbot P, Baltz JM. Cell volume regulation is initiated in mouse oocytes after ovulation. Development. 2009;136:2247–54. 17. Rosenbusch B, Hancke K.  Conjoined human oocytes observed during assisted reproduction: description of three cases and review of the literature. Rom J Morphol Embryol. 2012;53:189–92. 18. Ebner T, Shebl O, Moser M, Sommergruber M, Tews G. Developmental fate of ovoid oocytes. Hum Reprod. 2008b;23:62–6. 19. Balakier H, Bouman D, Sojecki A, Librach C, Squire JA. Morphological and cytogenetic analysis of human giant oocytes and giant embryos. Hum Reprod. 2002;17:2394–401. 20. Machtinger R, Politch JA, Hornstein MD, Ginsburg ES, Racowsky C.  A giant oocyte in a cohort of retrieved oocytes: does it have any effect on the in vitro fertilization cycle outcome? Fertil Steril. 2011;95(2):573–6. 21. Rama Raju GA, Prakash GJ, Krishna KM, Madan K. Meiotic spindle and zona pellucida characteristics as predictors of embryonic development: a preliminary study using PolScope imaging. Reprod Biomed Online. 2007;14:166–74. 22. Sousa M, Teixeira da Silva J, Silva J, Cunha M, Viana P, Oliveira E, Sá R, et al. Embryological, clinical and ultrastructural study of human oocytes presenting indented zona pellucida. Zygote. 2015;23(1):145–57. 23. Farhi J, Nahum H, Weissman A, Zahalka N, Glezerman M, Levran D. Coarse granulation in the perivitelline space and IVF-ICSI outcome. J Assist Reprod Genet. 2002;19:545–9. 24. Hassan-Ali H, Hisham-Saleh A, El-Gezeiry D, Baghdady I, Ismaeil I, Mandelbaum J.  Perivitelline space granularity: a sign of human menopausal gonadotrophin overdose in intracytoplasmic sperm injection. Hum Reprod. 1998;13:3425–30. 25. Verlinsky Y, Lerner S, Illkevitch N, Kuznetsov V, Kuznetsov I, Cieslak J, Kuliev A. Is there any predictive value of first polar body morphology for embryo genotype or developmental potential? Reprod Biomed Online. 2003;7:336–41. 26. Navarro PA, de Araujo MM, de Araujo CM, Rocha M, dos Reis R, Martins W. Relationship between first polar body morphology before intracytoplasmic sperm injection and fertilization rate, cleavage rate, and embryo quality. Int J Gynaecol Obstet. 2009;104:226–9.

Semen Analysis Stuart Benjamin John Dawe-Long

Basic laboratory equipment needed is given in Fig. 1 and described in Table 1. Fig. 1  Basic laboratory equipment for semen analysis

S. B. J. Dawe-Long (*) Andrology Laboratories, Manchester University NHS Foundation Trust, Manchester, UK e-mail:[email protected] © Springer Nature Singapore Pte Ltd. 2023 S. Ghumman (ed.), Atlas of Assisted Reproductive Technologies,



S. B. J. Dawe-Long

Table 1  Equipment, consumables, and reagents required for semen analysis Examination stage Equipment required Pre-examination Balance Incubator Macroscopic Microscopic

Consumables required Reagents required

Analysis Volume To reduce viscosity and aid in liquefaction pH assessment Viscosity assessment Motility assessment Concentration assessment Morphology assessment Vitality assessment Round cell assessment

pH strips/meter Pasteur pipette Heated stage Vortex mixer Microscope (bright-field and phase contrast capability) Air displacement pipettes (ADP) Positive displacement pipettes (PDP) Multi-cell counter Improved Neubauer haemocytometer (INH) Glass slides, coverslips, microtubes, pipette tips

Diluent, staining reagents for morphology, i.e. rapid romanowsky, vitality stain

Semen Analysis

Analysis should occur as below:



S. B. J. Dawe-Long

Step 1. Volume estimation. Weigh the semen sample and container. Subtract the empty container weight from this to get the volume of semen produced. The example from the photograph: 20.20 g–15.60 g = 4.6 g or 4.6 mL.

Step 2. Assess viscosity/liquefaction. This is undertaken using a wide bore pipette and measuring bead length of the semen as it drops from the tip. If >2 cm, this is classed as viscous. Liquefaction is assessed by reviewing whether streaks/ gelatinous bodies are present after 60 min.

Semen Analysis


Step 3. Measure the pH. Simple test strips can be used. Following step 2, use a drop on the indicator strip, leave for a minute for the colour to stabilise and assess. In this example, the pH given is 8.0.

Step 4. Prepare slide for motility. A glass slide is placed onto a heated stage (36 °C). Place 2 × 10 μL drops of well-mixed semen onto the slide using a PDP. Place a warmed coverslip over each drop and leave for 60 seconds to settle.

Step 5. Assess motility. Sperm can be placed into four categories for movement. Grade ‘A’ sperm move in a linear motion at a speed of about 1/2 a tail length per second. Grade ‘B’ sperm move linear, between one head length and less than 1/2 a tail length per second. Grade ‘C’ sperm are moving but may not be linear and may move extremely slow. Grade ‘D’ sperm are completely immotile. Categorise at least 200 sperm in two separate aliquots and calculate the average of each grade. If the motility is reduced below the lower reference limit, consider undertaking a viability test.

S. B. J. Dawe-Long


Step 6. Preparation of dilution tubes for concentration. Assess the dilution factor by estimating the numbers of sperm in each field (see Table 2). Place the diluent into the tubes using an ADP and an excellent pipetting technique.

Table 2  Concentration dilution estimations [1, 2] Sperm per ×40 field 1–15 16–40 41–200 >200

Dilution 1 + 4 (1:5) 1 + 9 (1:10) 1 + 19 (1:20) 1 + 49 (1:50)

Diluent (μL) 200 450 950 2450

Well-mixed semen (μL) 50 50 50 50

Semen Analysis


Step 7. Addition of semen to diluent for concentration. Ensure a PDP is used to add the semen into the diluent. Vortex each tube for 10–15 s.

Step 8. Preparation of the INH for concentration. Use a special haemocytometer coverslip to fix onto an INH with a small amount of moisture. Take 10 μL of each dilution mixture and load each side of the chamber. Leave this to settle for at least 10 min in a humid chamber.

Step 9. Assess the INH and calculate concentration. Under phase-contrast microscopy, assess each side of the INH. The aim is to count 200 sperm in complete rows (the red arrows show the direction of counting). If 200 sperm are reached before you reach the end of a row, continue to the end. If they are not counted at the end of each row, continue to the next as shown by the arrow [1, 2]. Total numbers


S. B. J. Dawe-Long

are then divided by an appropriate division factor from the WHO guidelines [1]. Example, if two rows on each side of the chamber gave a total of 422 sperm, a division of 4 is applied to give a concentration of 105.5 million sperm/ml. (i)



Semen Analysis


Step 9 (i–iii). Preparation of morphology. Place 10 μL drop of semen onto the bottom of a labelled slide using a PDP (i). Take a second slide and place this flush against the slide above the drop of semen. Gently pull the slide back, dragging the semen with it (ii). Push the slide forward retaining gently pressure in a steady motion (iii). Leave to air-dry before following with your fixing/staining procedure (example in Step 10).

Step 10. Staining morphology slides. The precise method of staining will depend on your chosen technique. A simple rapid staining method can be used. The three items shown are: solution A (methanol fixative), solution B (Eosin Y