Syllabus: 

COURSE DIRECTOR: Gianpiero Palermo

ASSISTANT DIRECTORS: Doris Baker,               

CONTRIBUTING FACULTY: Dmitri Dozortsev, Queenie Neri, Michael Reed, Sherman Silber, Peter Nagy, Andrew Rybouchkin, Ryuzo Yanagimachi

Course Name: Assisted Fertilization  – 2 CEUs

Prerequisite: A minimum of BS in biology, biomedical sciences, reproductive sciences or reproductive physiology. 

Examination site: http://www.embryologyeducation.org/index.htm

Grading:

Assisted Fertilization Level IV – – Embryologist (EMB): Ph.D., M.D., OR non-doctoral meeting grandfathering clause in 2011

Course Description: The course will include a review of the history of gamete micromanipulation, mechanism of fertilization,  mechanism of parthenogenetic activation, protocols for parthenogenetic activation and activation factor deficiency, late fertilization, abnormal fertilization, Kartagener's syndrome, globospermia, and other causes of failed fertilization., in-vivo and in vitro aging of the sperm cells, technical errors encountered when performing ICSI, rescue ICSI, identification of spermatozoa, sperm precursors and other testicular cells in wet preparation from  a testicular biopsy, and methods for selecting spermatozoa in testicular sample.  

Course Objectives:

By the end of the course, the participant will demonstrate that he/she will/can:

• Fully describe the mechanism of fertilization and discuss reasons for fertilization failure. 
• Describe types of oocyte culture media.
• Define oocyte activation and describe molecular processes involved.
• Define pathogenic activation and explain how it may occur.
• Fully describe the molecular and chromosomal events taking place before and after fertilization   
• Outline the history leading up to the first intracytoplasmic sperm injection (ICSI)  procedure.
• Give indications for ICSI.
• Describe chromosomal errors and subsequent genetic outcomes associated with the ICSI procedure.
• Explain the mechanism of oocytes aging and its impact on subsequent fertilization and activation outcomes.
• Describe the ageing process of sperm cells and select the optimal sperm collection protocol.
• Determine the optimal time window for sperm injection.
• Identify spermatogenic cells in the mount preparation from a  TESA/TESE sample and apply the protocol for the optimal recovery of the sperm cells.  
• List technical errors to avoid while performing ICSI.
• Apply protocols of artificial activation in cases of activation factor deficiency in human spermatozoa.
• Discuss metabolism of ooyctes through early cleavage stage embryos.

 

References and supplemental material

Book chapter:

Palermo GD, Neri QV, Takeuchi T, Hong SJ, Rosenwaks Z: Intracytoplasmic Sperm Injection: Technical Aspects. In Gardner DK, Weissman A, Howles CM, Shoham Z (eds): "A Comprehensive Textbook of Assisted Reproductive Technology: Laboratory and Clinical Perspectives, Third Edition". Martin Dunitz LTD, London, England, 2009, pp. 171-180

Manuscripts and supplemetal material: 

History

Uehara T, Yanagimachi R. Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol Reprod. 1976 Nov;15(4):467-70 Lanzendorf SE, Maloney MK, Veeck LL, Slusser J, Hodgen GD, Rosenwaks Z. A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes. Fertil Steril. 1988 May;49(5):835-42. Fishel S, Jackson P, Antinori S, Johnson J, Grossi S, Versaci C. Subzonal insemination for the alleviation of infertility. Fertil Steril. 1990 Nov;54(5):828-35. Cohen J, Talansky BE, Malter H, Alikani M, Adler A, Reing A, Berkeley A, Graf M, Davis O, Liu H, et al. Hum Reprod. 1991 Jan;6(1):118-23. Microsurgical fertilization and teratozoospermia.

The first ICSI pregnancy

Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Lancet. 1992 Jul 4;340(8810):17-8. ICSI technique modifications: Nagy ZP, Oliveira SA, Abdelmassih V, Abdelmassih R Novel use of laser to assist ICSI for patients with fragile oocytes: a case report. Reprod Biomed Online. 2002 Jan-Feb;4(1):27-31.

Rescue ICSI

Nagy ZP, Joris H, Liu J, Staessen C, Devroey P, Van Steirteghem AC.Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes. Hum Reprod. 1993 Dec;8(12):2180-4. Morton PC, Yoder CS, Tucker MJ, Wright G, Brockman WD, Kort HI.Reinsemination by intracytoplasmic sperm injection of 1-day-old oocytes after complete conventional fertilization failure. Fertil Steril. 1997 Sep;68(3):488-91.

Mechanism of ICSI

Sperm plasma membrane damage

The mechanism of fertilization after ICSI is markedly different from that during natural fertilization. Whereas during normal fertilization process, sperm-oocyte fusion is followed by incorporation in the cytoplasm of a demembranated, "naked" sperm nucleus, which immediately becomes accessible for ooplasmic factors, especially thiol-reducing agents (Perreault et al., 1984), following ICSI not a naked sperm nucleus but the whole sperm cell, enclosed in i ts membranes, is exposed to the ooplasm (Palermo et al., 1993, Van Steirteghem et al., 1993). It has been demonstrated that whole sperm cells behave in a different way than partially demembranated sperm cells in cytoplasmic cell extracts (Maleszewski, 1990). If an intact spermatozoon injected into the ooplasm, there will be no interaction between the spermatozoon and the oocyte. This because ooplasm does not have an enzyme to digest sperm plasm membrane to enable interaction. Therefore, sperm plasma membrane has to be damaged prior to the fertilization. Even though sperm cell appear to have distinctive compartments, such as a head, mid piece and the tail, it has only one plasma membrane covering the entire cell. Therefore, sperm plasma membrane can be damaged in any area, in order to enable sperm-egg interaction after injection. Usually, during ICSI, the sperm plasma membrane is damaged by “touching” tail, which in reality is pressing the sperm tail with a glass pipette, until the sperm membrane damage becomes sufficient for the membrane to leak and loose its ability to maintain cell integrity, which leads to disabling Na/K pump and loosing membrane potential. To the observer this event is marked by the sperm ceasing its movement. The sperm membrane damage can also be revealed using vital staining technique, such as Eosin B. Vital staining is based on the intact membrane ability to exclude Eosin B. But once the membrane loses its charge, Eosin B migrates inside of the cell and stains the nucleus. 

Effect of PVP

 

Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation.

Dozortsev D, Rybouchkin A, De Sutter P, Dhont M.

 

PVP, which is usually present in the culture medium has the ability to act as a membrane’s “band-aid”, and prevents the stain migration into the nucleus .

In the image above, sperm cells immobilized by touching “touching tail” in the presence of Eosin B, do not uptake the stain in the presence of PVP However, once PVP is washed-out, the membrane damage becolow. (Images above and below from Dozortsev et al, 1995, Human Reproduction, reference in the text). 

 

Sperm after immobilization in the presence of Eosín B and PVP, transferred into the medium with Eosin B, without PVP. A. Sperm on the right immediately after the transfer. B. 1 min after the transfer. C. 3 minutes after the transfer. Sperm on the left is a stained control. The plasma membrane damage induced by “touching” tail does not allow anything from the sperm cell to “leak” into surrounding medium. Indeed, the sperm cells retain full capacity to fertilize an oocyte for several hours following immobilization.

Initial sperm nucleus swelling 

Within minutes after injection, the sperm nucleus becomes accessible the thiol reducing agents of the ooplasm, and swells.

(Images in this section are from Dozortsev et al, 1995, Human Reproduction)

Swollen sperm nucleus 20 minutes after injection into 1 day old human oocyte. The same process precedes pronucleus formation after normal fertilization.

It is important to understand that this initial swelling is not under the same control as nuclear decondensation necessary for pronuclei formation. This swelling can also be easily duplicated in vitro using thiol reducing, such as DTT.

Decondenced human sperm nucleus after sperm immobilization in the presence of DTT (top). Intact sperm nuclei after sperm immobilization during incubation in lhe simultaneous presence of bath DTT and PVP (bottom).

The most abundant low-molecular-weight non-protein thiol, exists in two forms: reduced GSH and GSH disulfide (GSSG). It has been clearly shown that GSH is a disulfide bond reducer present in the cytoplasm of oocytes (Perreault et al. 1984, 1988). Oocytes can synthesize GSH (tripeptide -glutamyl-cysteinyl-glycine; GSH) during the first meiosis. Oocyte-derived GSH seems to assure the reduction of disulfide bonds in sperm nucleus. This, in turn, promotes nuclear decondensation for male pronucleus formation during fertilization (reviewed by Sutovsky & Schatten 1997). Thus, GSH provides the reducing power to initiate chromatin decondensation, prior to the male pronucleus formation (Yoshida et al. 1993, Funahashi et al. 1994). Depletion of endogenous GSH by a specific inhibitor of GSH synthesis during bovine oocyte maturation blocks the formation of a male pronucleus and prevents the assembly of sperm aster microtubules (Sutovsky & Schatten 1997). The elevated levels of oocyte GSH can enhance male pronuclear formation after IVF (Funahashi et al. 1994). If an excessive amount of PVP is injected with the sperm cell, for the reasons described above, sperm and egg interaction can be delayed.

Sperm Associated Oocyte Activating factor release

The initial swelling of the sperm nucleus ruptures sperm plasma membrane and enables the release of the sperm-associated, oocyte-activating factor - PLC zeta - into the ooplasm. The entire mechanism of fertilization after ICSI can be summarized as the following:

1. Sperm plasma membrane damage is induced by “touching” tail, but the damage is masked as long as PVP is present.
2. Following injection, PVP is diluted and reduced GSH and GSH disulfide (GSSG ) enter the sperm nucleus inducing sperm nucleus swelling within a few minutes
3. The nuclear swelling ruptures sperm plasma membrane, enabling the contact between nucleus bound PLC zeta and ooplasm, inducing oocyte activation and leading to chromatin decondensation.

 

Human oocyte activation following intracytoplasmic injection: the role of the sperm cell. Dozortsev D, Rybouchkin A, De Sutter P, Qian C, Dhont M.

Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction. Dozortsev D, Qian C, Ermilov A, Rybouchkin A, De Sutter P, Dhont M.

 

Timing of nuclear progression after ICSI

(Images in this section are fro Dozortsev et al, 1995, Human Reproduction)

Intact metaphase plate in 1 day old oocyte

This 1 day old human oocyte was fixed within a few minutes after sperm injection. Note that some chromatids began to separate. This separation may be the result of activation    (more likely) or, may represent an artefact of ageing.

Early telophase about 4.5 hrs after injection. Note that at this stage, the sperm nucleus does not decondense, but remains swollen. Also note that oocyte and polar body chromosomes forming two groups are indistinguishable from each other.

 

Late telophase (aka chromatin mass stage)shortly after 2PB extrusion. Sperm chromatin remains swollen, while oocyte chromosomes are tightly packed into the mass, 

  

Pronuclei formation. At this stage the pronuclei cannot be seen under the microscope, because the visible part, nuclear membrane has not been formed yet. Note the striking difference between male and female pronuclei.  

 

This oocyte was displaying a single pronucleus the day after ICSI. The sperm head is swollen, indicating that it was correctly injected and has interacted with an oocyte resulting in activation. However, it failed to decondense for unknown reasons. The resulting haploid embryo will not be viable. 

 

In this instance decondensing sperm chromatin can be seen entangled with forming female pronucleus. The result is likely a single pronucleated diploid zygote. Such embryos have been shown to implant and result in term pregnancy.  

Stice SL, Robl J M. Activation of mammalian oocytes by a factor obtained from rabbit sperm. Mol Reprod Dev. 1990 Mar;25(3):272-80. Tesarik J, Sousa M, Testart J. Human oocyte activation after intracytoplasmic sperm injection. Hum Reprod. 1994 Mar;9(3):511-8. Dozortsev D, Rybouchkin A, De Sutter P, Qian C, Dhont M. Human oocyte activation following intracytoplasmic injection: the role of the sperm cell. Hum Reprod. 1995 Feb;10(2):403-7. Dozortsev D, Rybouchkin A, De Sutter P, Dhont M. Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation. Hum Reprod. 1995 Nov;10(11):2960-4. Parrington J, Swann K, Shevchenko VI, Sesay AK, Lai FA. Calcium oscillations in mammalian eggs triggered by a soluble sperm protein. Nature. 1996 Jan 25;379(6563):364-8. Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM,  K, Lai FA. PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development. Development. 2002 Aug;129(15):3533-44.

Optimal Fertilization window 

 

Dozortsev D, Nagy P, Abdelmassih S, Oliveira F, Brasil A, Abdelmassih V, Diamond M, Abdelmassih R. The optimal time for intracytoplasmic sperm injection in the human is from 37 to 41 hours after administration of human chorionic gonadotropin. Fertil Steril. 2004 Dec;82(6):1492-6.

 

Fertilization failure

References:

Rybouchkin AV, Van der Straeten F, Quatacker J, De Sutter P, Dhont M. Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity. Fertil Steril. 1997 Dec;68(6):1144-7. Murase Y, Araki Y, Mizuno S, Kawaguchi C, Naito M, Yoshizawa M, Araki Y. Pregnancy following chemical activation of oocytes in a couple with repeated failure of fertilization using ICSI: case report. Hum Reprod. 2004 Jul;19(7):1604-7. Epub 2004 Apr 29. Kyono K, Kumagai S, Nishinaka C, Nakajo Y, Uto H, Toya M, Sugawara J, Araki Y. Birth and follow-up of babies born following ICSI using SrCl2 oocyte activation. Reprod Biomed Online. 2008 Jul;17(1):53-8. Egashira A, Murakami M, Haigo K, Horiuchi T, Kuramoto T. A successful pregnancy and live birth after intracytoplasmic sperm injection with globozoospermic sperm and electrical oocyte activation. Fertil Steril. 2009 Dec;92(6):2037.e5-9. Epub 2009 Oct 1. Nasr-Esfahani MH, Deemeh MR, Tavalaee M. Artificial oocyte activation and intracytoplasmic sperm injection. Fertil Steril. 2009 Apr 24.

Experimental and theoretical basis for artificial activation of human oocytes

Mechanisms of activation failure

Case when all MII oocytes become fertilized following ICSI are not common. In fact, the average fertilization rate after ICSI is usually below 80%. Cytological analysis of oocytes that failed fertilized usually reveals that spermatozoon was interacting with the ooplasm, but failed to trigger the activation (Dozortsev et al., 1994).

Recently ovulated human oocytes are extremely resilient to parthenogenetic activation by sham-ICSI (Dozortsev et al, 1995). Therefore, even if fertilization rate is very low, it is usually due to the presence of some activating factor in the spermatozoon. Thus, the true deficiency of the activating factor should only be suspected, when less than 10% of the oocytes fertilized.

In most cases failure of fertilization can be forecasted based on sperm morphology. It is very uncommon that a morphologically normal motile (live) spermatozoon fails to activate an oocyte due to activating factor deficiency. If activation fails in such case, the activation problem is most likely due to the oocyte.

At the same time, it is important to understand that morphological assessment of the sperm cells is not predictive of its activating potential.

For example, acrosomeless (globospermic) spermatozoa in some cases will successfully activate human oocytes (Dirican et al., 2008, Stone et al., 2000), while in others their injection will result in 100% failure of activation (Heindryckx et al, 2005). This may to certain extent correlate with acrosine positive or acrosine negative status of acrosomeless spermatozoa, which in its own turn seem to correlate with the respectively presence of absence of PLC zeta.

Similarly, in the case of Kartagener's syndrome, immotile spermatozoa may still activate an oocyte (Matsumoto et all, 2010).

Since there are strong evidences that PLC zeta is the sperm-derived activating factor, one could argue that using respective antibodies would provide a discriminating testing. However, due to morphological peculiarities of the sperm cell, in situ antibodies testing may often be non-conclusive.

Therefore, functional testing, by injecting spermatozoa in question into the oocytes seem to be the most feasible assay at this time (Heindryckx et al, 2005).

Tests for sperm activating ability

Because sperm activating factor (PLC –zeta) is not species-specific (Dozortsev et al, 1995, Rybouchkin et al, 1996; Terada et al, 2004) mouse, bovine as well as oocytes of other mammals oocytes can be used to test activating ability of human spermatozoa.

Hamster eggs usually survive injection better and they can be purchased frozen. However, they are  expensive and may be activated by pricking itself, masking true activation capacity of a spermatozoon (although we were not able to activate by sham-ICSI any of the frozen-thawed hamster’s eggs).  Also, importantly, there is no clear benchmark activation rate has been established for activating potential of the human spermatozoa using hamster’ eggs. 

Mouse eggs on the other hand, don’t survive injection as well as hamster eggs and we were no able to locate the source of frozen mouse eggs. Similarly to hamster, mouse eggs may also be activated by pricking, particularly as they age and their sensitivity to parthenogenetic stimuli increases.

Therefore, it is recommended that for the purpose of sperm activation testing mice would be sacrificed shortly after expected ovulation time and used within 4 hrs after expected ovulation.

One clear advantage of mouse eggs is that their activation rate (excluding eggs which are damaged or were not injected correctly) following injection of normal human spermatozoa is 100%.

Therefore, any deviation from this benchmark is strongly suggestive of activation factor deficiency.

Even though the functional tests are available, their practical significance is not very high, because usually, the initial ICSI attempt will serve as a test and as experience demonstrates, the underlying reason of failed fertilization will not affect the subsequent treatment modality. 

Artificial activating methods

Development can be set into motion by a number of artificial stimuli, although their range is smaller than in mice.  For example, ethyl alcohol, a very potent activator of mouse oocytes, is not able to induce activation of human oocytes, even 1 day old (Dozortsev et al, unpublished).

Majority of physical and chemical activating stimuli used for artificial activation in the humans, elicit Ca++ release into the cytoplasm, mimicking to different extent Ca++ release taking place during natural fertilization (Nasr-Esfahani et al, 2009).

The end-point of activation is a physical destruction of cyclin B, which leads to inactivation of p34 kinase and drop in MPF activity (Hyslop et al, 2004) . 

Even though animal research and in particular Ozil’s work (1990) assign the importance to the pattern of the Ca++ oscillations during artificial activation, the relevance of activation patter for human oocytes injected with the sperm is not certain.

This is because in animals, the impact of artificial activation stimuli have been tested largely on parthenogenetic embryos, which do not generally develop well due imprinting problems. In fact, in mammals, even when an oocyte is activated by sperm, but the male genome does not participate in development (gynogenesis), embryo development is usually poor and rarely survive far past implantation. 

Cloning experiments, where only a tiny minority of embryos develop to term (Wakayama and yanagimachi, 2001) also illustrates an overwhelming importance of genetic makeup of the embryo over the activation modality.

One of the potent artificial activating agents of human oocytes – Puromycin – has to mentioned separately. This is because it does not cause Ca++ release (Dozortsev, unpublished) and probably acts by suppressing synthesis of the cyclin B and by inhibiting its phosphorylation, rather than by its physical destruction.  The point of no return in Puromycin activated oocytes is most likely the initiation of DNA synthesis (Dozortsev, unpublished).

This observation has practical importance, because it predicts that Puromycin will act synergistically with Ca++ engaging activation stimuli.

Indications for artificial oocyte activation

Based on the current experience, fertilization failure of any ethyology is an indication for artificial activation. 

Also, it is important to note that even though oocytes that failed to become fertilized following ICSI can be successfully activated on day 1 and many display 2 pronuclei and a second polar body, no pregnancy has been reported.

Therefore, only fresh cases are the candidates for the procedure. 

Rescue of failed ICSI are likely to be futile not solely because of oocyte ageing, since rescue ICSI following conventional insemination has been reported to result in pregnancy.

The confounding factor for rescue activation is probably sperm chromatin deterioration to the extent that prevents embryonic development.

 

Protocols resulted in live birth after artificial activation:

Considerations:

As a general rule, activation stimulus has to be applied to the oocytes at the same time or shortly after sperm injection. If activation stimulus is applied before sperm injection, changes in the cytoskeleton and plasma membrane may make oocytes more fragile and prone to damage. On the other hand, in the absence of an activation stimulus, injected sperm chromosomes quickly undergo premature chromosomes condensation and the significant delay between injection and activation may lead to the loss of sperm chromosomes (accessory micronuclei, along side of the pronuclei may be seen in such case).  

Another practical consideration is the timing of ICSI and activation relative to hCG administration. Oocytes generally become more susceptible to parthenogenetic activation as they age. However, it has been shown that optimal fertilization window is between 39 and 42 hrs after hCG (Dozortsev et al, 2004). Therefore, it would seem that the best time for artificial activation would be around 42 hrs after hCG.

It should also be noted that if oocyte deficiency is suspected as a underlying reason of failed fertilization, delaying ICSI and activation until 45-47 hrs post hCG may be desirable. 

Specific protocols:

First author/reference/contact: Rybouchkin et al., 1997; andrei.rybouchkin@rug.ac.be

Diagnosis: Globozoospermia

Outcome: Ongoing pregnancy

Protocol:  

Four different procedures for assisted oocyte activation were applied to the donated oocytes:

1) 111 vigorous aspiration of oocyte cytoplasm during sperm injection (6)

2) 121 same as above protocol plus ionophore A23187 treatment (5 µM during 7 minutes) at 30 minutes after sperm injection

3) 131 injection of approximately 5pl of O.lM CaClz along with a spermatozoon, followed by ionophore treatment at 30 minutes after ICSI ( This procedure has been propose by this study.)

4)141 the same as above plus with ionophore treatment 30 and 60 minutes after injection.

First author/reference/contact: Kim et al., 2001; stkim@kyuh.co.kr

Diagnosis:  Round-headed spermatozoa 

Outcome:  Fertilization rate, implantation, pregnancy, and delivery.

[21 of 35, 60%; 2 pronuclei in 18 of 21; 3 pronuclei in 3 of 21]

Protocol:

Oocyte aspiration was performed with transvaginal ultrasound guidance. After 5 hours, all oocytes were denuded enzymatically with 0.1% hyaluronidase (Sigma, St. Louis, MO) for 30–60 seconds, followed by mechanical denudation. Motile round-headed spermatozoa were injected into oocytes in metaphase II, and assisted oocyte activation was performed with calcium ionophore A23187 (Sigma). At approximately 16–18 hours after injection, the presence of 2PN was recorded as a sign of fertilization. 

First author/reference/contact: Eldar-Geva, et al., 2003; gevat@szmc.org.il

Diagnosis: Normozoospermic patient with previous repeated failed fertilization after ICSI (Case report)

Outcome: Fertilization rates in three cycles were 4/6, 5/16 and 7/20 oocytes. Two pregnancies were achieved; the first ended with second trimester miscarriage due to fetal anomaly and the second with a delivery of three healthy babies.

Protocol:

Within 1 hour of injection, 6 oocytes were exposed to 10 µmol/L of ionophore A23187 inIVF-50 medium for 7 minutes at 37°C in 5%CO2. The oocytes were then washed free of the ionophore through 10 drops of fresh culture medium and incubated further as usual.

First author/reference/contact: Chi et al., 2004; hanna129@hanmail.net

Diagnosis: Normozoospermic patient (Case report)

Outcome: The fertilization rate of oocytes activated (12 of 15, 80.0%) was higher than that of the nonactivated oocytes (4 of 16, 25.0%). Twin pregnancy.

Protocol:

Thirty minutes after ICSI, the oocytes were exposed to 8 µmol/L calcium ionophore for 8 minutes and subsequently washed thoroughly in P1 medium

 

First author/reference/contact: Heindryckx et al., 2008; bjorn.heindryckx@UGent.be

Diagnosis: Failed or low fertilization in previous ICSI cycles or who had well-known sperm-borne activation deficiencies such as globozoospermia.

Outcome: High fertilization and acceptable pregnancy rates.

Protocol:

Oocytes were kept at 37°C in a 6% CO2 air atmosphere in Cook Cleavage medium (Cook Ireland Ltd, Limerick, Ireland).  For ICSI with AOA, spermatozoa resuspended in HEPES-buffered Oocyte Wash (Cook Ireland Ltd) and an equal volume of 8% polyvinylpyrrolidone (PVP ICSI-100, VitroLife Sweden AB, Kungsbacka, Sweden) was immobilized by pressing the tail to the bottom of the dish and was drawn up into an injection pipette and the sperm head was kept at the very tip of the pipette. Then the pipette was moved to a drop of 0.1 mol/l CaCl2 and an amount of CaCl2 was aspirated into the injection pipette, which corresponded to the diameter of the oocyte. Oocytes were conventionally injected with the spermatozoa and CaCl2 and kept in Cook Cleavage medium for 30 min. Injected oocytes were exposed for 10 min in the incubator to 10 μmol/l Ca2+ ionophore (Ionomycin, cat. no. 159611; MP Biomedicals) dissolved in Cook Cleavage medium and subsequently washed intensively and put in Cook Cleavage for 30 min in the incubator. Finally, ionophore treatment was repeated during 10 min and after intensive washing, oocytes were placed in Cook Cleavage medium for culture. 

 

First author/reference/contact: Nasr-Esfahani et al., 2008; mh_nasr@med.mui.ac.ir

Diagnosis: Severe teratozoospermia 

Outcome: Improvement of fertilization and cleavage rates after AOA

Protocol:

Oocytes were randomly divided into two groups: control and AOA. The injected oocytes in the control group were cultured in G1. The remaining oocytes were chemically activated by exposure to 10 mM Ionomycin for 10 minutes.

First author/reference/contact: Kyono et al. 2008; info@ivf-kyono.or.jp

Diagnosis: Oocytes from patients with repeated fertilization failure

Outcome: Live birth  

Protocol:

Materials

SrCl₂･6H₂O :SIGMA-ALDRICH #255521, Dolbecco’s Modified Eagle Medium（DMEM : GIBCO #21068

Method

1.                  SrCl₂･6H₂O Stock solution. We prepared 100mM SrCl₂ stock solution in DMEM, and stocked at -30℃.

_2.                    The micro droplet of 10mM SrCl₂ solution (in DMEM) was overlaid mineraloil, and stored in 6%Co₂, 5%O₂, 89.9%N air condition overnight.

 

 

Provided by Koichi Kyono, M.D., Ph.D

These oocytes were cultured in Universal IVF Medium for 30 min and then activated in SrCl2 (10 mmol/l), 10% synthetic serum supplement, and Dulbecco’s modified Eagle’s medium (Gibco, USA) 20 μl/drop under 6% CO2, 5% O2, and 89% N2 under humidified conditions for 60 min.

 

First author/reference/contact: Ahmady et al., 2007; ali_ahmady@yahoo.com

Diagnosis: nonviable testicular sperm is used for intracytoplasmic injection (ICSI) 

Outcome: full term delivery  

Protocol:

The injected oocyte was incubated in G-Fert (Vitrolife, Englewood, Colo) medium containing 10 mg/mL calcium ionophore A23187 (stock solution 10 mg/mL in dimethyl sulfoxide A23187 (stock solution 10 mg/mL in dimethyl sulfoxide stored at 220C; Sigma Chemical Co, St Louis, Mo) for10 minutes.  

First author/reference/contact: Heindryckx et al., 2005; Bjorn.Heindryckx@UGent.be

Diagnosis: Patients with previously failed fertilization and Globozoospermia 

Outcome: After AOA, fertilization rates were 77 and 71% in the sperm- and oocyte-related groups respectively. Five pregnancies were achieved in the globozoospermia group and three in cases of oocyte-related activation failure.

Protocol:

Activation capacity was assessed by 2-cell formation (mouse oocyte activation test, MOAT). When no activation occurred, AOA was done by ICSI with CaCl2 followed by a Ca21 (0.1 mol/l) ionophore (10 mmol/l ionophore) exposure.

 

First author/reference/contact: Juan Chen et al., 2010; qianyun@njmu.edu.cn

Diagnosis: Patients with fertilization failure or low fertilization rates 

Outcome: Improve fertilization rates (78.8%) and embryo quality (41.5% (17/41) in cases with fertilization failure after ICSI.

Protocol:

Oocytes were activated in calcium-free HTF medium containing 10% (v/v) SSS and 10mM SrCl2 (Sigma Chemical) for 60 min at 37◦C and 5% CO2 (Yanagida et al. 2006).

 

ICSI and oocytes dysmorphism

Alikani M, Palermo G, Adler A, Bertoli M, Blake M, Cohen J. Intracytoplasmic sperm injection in dysmorphic human oocytes. Zygote. 1995 Nov;3(4):283-8.

 

ICSI with non-ejaculated spermatozoa

Recognizing cells in the testicular sample

Devroey P, Liu J, Nagy Z, Tournaye H, Silber SJ, Van Steirteghem AC. Normal fertilization of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection. Fertil Steril. 1994 Sep;62(3):639-41. Silber SJ, Nagy ZP, Liu J, Godoy H, Devroey P, Van Steirteghem AC. Conventional in-vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration. Hum Reprod. 1994 Sep;9(9):1705-9. Tournaye H, Liu J, Nagy PZ, Camus M, Goossens A, Silber S, Van Steirteghem AC, Devroey P. Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular spermatozoa. Hum Reprod. 1996 Jan;11(1):127-32. Silber SJ, Johnson L. Are spermatid injections of any clinical value? ROSNI and ROSI revisited. Round spermatid nucleus injection and round spermatid injection. Hum Reprod. 1998 Mar;13(3):509-15. Tesarik J Oocyte activation after intracytoplasmic injection of mature and immature sperm cells. Hum Reprod. 1998 Apr;13 Suppl 1:117-27. Ramasamy R, Lin K, Gosden LV, Rosenwaks Z, Palermo GD, Schlegel PN. High serum FSH levels in men with nonobstructive azoospermia does not affect success of microdissection testicular sperm extraction. Fertil Steril. 2009 Aug;92(2):590-3. Epub 2008 Oct 29.

Spermatozoa-affecting syndromes and ICSI

Bourne H, Richings N, Harari O, Watkins W, Speirs AL, Johnston WI, Baker HW. The use of intracytoplasmic sperm injection for the treatment of severe and extreme male infertility. Reprod Fertil Dev. 1995;7(2):237-45 von Zumbusch A, Fiedler K, Mayerhofer A, Jessberger B, Ring J, Vogt HJ. Birth of healthy children after intracytoplasmic sperm injection in two couples with male Kartagener's syndrome. Fertil Steril. 1998 Oct;70(4):643-6. Abu-Musa A, Hannoun A, Khabbaz A, Devroey P. Failure of fertilization after intracytoplasmic sperm injection in a patient with Kartagener's syndrome and totally immotile spermatozoa: case report. Hum Reprod. 1999 Oct;14(10):2517-8 Dam AH, Feenstra I, Westphal JR, Ramos L, van Golde RJ, Kremer JA. Globozoospermia revisited. Hum Reprod Update. 2007 Jan-Feb;13(1):63-75. Epub 2006 Sep 28. Short tail sperm cells after vasectomy reference needed

Testicular biopsy and ICSI

Testicular biopsy, fine needle aspiration, and microdissection of individual tubules can yield motile, viable spermatozoa for ICSI.  Identification of viable sperm in a homogenized tubule preparation requires sorting through the multiple cell types common to testicular tubules to find the most appropriate sperm cell for injection.  Sperm cells are selected first for motility, and secondarily for morphology.  Testicular sperm are immature, and motility may be manifest as slow or rapid non-progressive twitching, and on occasion, motility with forward progression.  Though somewhat controversial, application of a motility stimulant, e.g. pentoxifylline may be required to induce motility, in fresh or cryopreserved specimens (Tasdemir et al, 1998; Terriou et al, 2000; Griveau et al, 2006; Kovacic et al, 2006).

Identification of sperm cells in the preparation is easier when sperm cell density, a reflection of spermatogenesis, is normal across the bulk of the testes tissue; however, there are instances when spermatogenesis not consistent across all tubules, where the tissue will exhibit a mosaic pattern of tubule morphology.  Individual tubules can be evaluated in this case during microdissection, or alternatively, the testicular tissue may be mapped, using a grid approach, by multiple fine needle aspirations to locate individual foci of spermatogenesis, after which the specific regions of spermatogenesis may be explored further.   As described by Schlegel, 1999, individual tubules that are more likely to have sperm-containing regions would be larger in diameter and opaque, compared to regions without active spermatogenesis, and there is a greater probability of spermatogenesis in tubules nearer to a blood supply (see also Chan and Schlegel, 2000).

 

Close up view of testicular tubules and blood supply to tubules; in situ

Preparation of testicular tissue

Testicular tissue can be retrieved on-site near to the IVF laboratory, or off-site and transported to the IVF laboratory.  Additionally, the tissue can be collected the same day as the oocyte retrieval, or prior to the oocyte retrieval and held in culture overnight for example (Schiewe et al, 2004, Wood et al, 2003), or cryopreserved and stored in liquid nitrogen until needed (Gil-Salom et al, 1996; Allan and Cotman, 1997; Friedler et al, 1997; Schiewe et al, 1997; Ben-Yosef et al, 1999; Huang et al, 2000; Schiewe et al, 2004; Verheyen et al, 2004; Giorgetti et al, 2005; Garg et al, 2008).

Testicular tubules can be processed using a variety of techniques.  Although time consuming, individual tubules can be ‘milked’, where tubules are visualized under a dissection microscope, and a non-cutting instrument, the rounded edge of a glass pipette or a glass slide, for example, is pushed down and long the length of the tubule, pushing the contents of the tubule out of one end, and repeating this process as needed.  Alternatively, bulk tubules can be homogenized, using needles and/or scalpel blades or using sterile mortar and pestle-style tissue homogenizers.  The homogenate may be collected in medium, and centrifuged to concentrate the tubule contents for examination.  Enzymatic application may increase the efficiency of the procedure, increasing the yield of viable spermatozoa in individuals that have reduced spermatogenesis (Crabbe et al, 1998).

Identification of mature spermatozoa for ICSI

With active spermatogenesis, the cells in the homogenate would be represented by sperm and non-sperm cell types, where the most readily identifiable cell types are mature spermatozoa and red blood cells that have contaminated the preparation.  IVF personnel are comfortable with mature spermatozoa morphology, and identification of these cells in a testicular tubule homogenate is accomplished by looking for sperm tails, which usually stand out as linear structures surrounded by round cell types and clumps of round cell types.  The eye can also be trained to detect motion, against a background of cells that are non-motile.  The motion may be from the sperm itself, or indirectly from the sperm cell bumping or pushing against other cells, or motion of larger cells clumps from of the immature spermatozoa while they are still attached to the cells.  Due to incomplete separation of the spermatids, that can be multi-tail spermatozoa in the tubule tissue, as there are in the ejaculate.  Rolling suspected, multi-tail sperm cells against the bottom of the dish with a micropipette can help to unravel the tails.  The morphology of the immature spermatozoa can be distinctly different from ejaculated cells.  Specifically, proximal droplets can be more pronounced, as retained cytoplasm has not yet been reduced.  Additionally, elongating spermatids, with full tail formation but where the head of the sperm is still encased in cytoplasm, can also be found.  And if there are no mature spermatozoa present, these elongating spermatids may be considered for ICSI (Schiewe et al, 1997).

 

Sertoli cell only diagnosis

 

Normal spermatogenesis

 

[video:http://www.youtube.com/watch?v=CcYypESeEz8]

Note the rotating cell, about 15 microns across, in the center. It has multiple microvilli. Found in testicular biopsy of a man with primary testicular failure. This is vasa efferentia or proximal caput epithelial cell. These cells have cilia which sweep the sperm into the epididymis from the testis.

Spermatogenesis and precursor cell types

Identifying, correctly, the various cell types representing early stages of spermatogenesis in a fresh preparation is more difficult.  When mature spermatozoa are not identified in a testicular preparation, earlier stages may be identified and considered for ICSI, but for most IVF personnel, this practice should be restricted to identification and use of elongating spermatids, where there is a visible, obvious sperm tail, either complete or in the early stages of formation.   The use of haploid spermatozoa precursor cells has been considered for use in humans (for review see Yanagimachi, 2005), however it is very difficult to distinguish between haploid round spermatids and diploid round precursor cells, and as such the American Society for Reproductive Medicine and the Society for Assisted Reproduction Practice Committees have stated in a joint report, that ROSNI (or ROSI) should only be considered under approved experimental protocols (ASRM, 2008).  For more information on identification of the various cells types present in a testicular preparation, there are two excellent papers that describe the stages of human spermatogenesis, and present photos of both fixed and living cells.  Both papers are freely available from the respective journal websites (Johnson et al, 1999, 2001).  See also Silber, et al, 1997).

Cryopreservation of testicular tissue – practical considerations

Cryopreservation of surgically-retrieved sperm (vas fluid, epididymal aspirates) or testicular tissue affords opportunities for more efficient scheduling for banking of sperm, as the surgical procedures can be done at the time of vasectomy, vasectomy-reversal, diagnostic testicular biopsy, or as planned prior to an IVF cycle.  Sperm recovered from the vas deferens or from the epididymis can be cryopreserved using the same procedures as for ejaculated sperm.  Testicular tissue may be processed, e.g. homogenized, washed with culture medium, and then cryopreserved, using the same procedures as for ejaculated sperm, or the tissue may be cryopreserved as intact tubules (Allan and Cotman, 1997).

The cryopreservation method used in this laboratory, for ejaculate sperm, epididymal aspirates, and rinses of the vas deferens is as follows:

Equipment:

Cryopreservation straws; 0.5ml

Centrifuge; 300-500g, as needed

Conical centrifuge tubes, as needed

Sterile pipettes, as needed

Cryopreservation medium (commercial) with glycerol as the cryoprotectant with egg yolk

Refrigerator (approximately 4C) with shelf space for horizontal aluminum cryo canes

Liquid nitrogen tank; room to hang specimen below the frost line of tank, and above liquid level

1. The specimen may be centrifuged to reduce the final volume, or to concentrate the specimen, as needed.  Centrifugation at 300-500g for 15 minutes is usually sufficient to produce a pellet.  Re-suspend the pellet in hepes-buffered room temperature medium, to one-half of the final volume desired for cryopreservation.

2. Add an equal volume of room temperature cryoprotectant (Freezing Medium – TYB with glycerol and gentamicin; Irvine Scientific, Irvine CA), drop-by-drop, to the re-suspended sperm pellet from step 1.  Mix gently, and transfer the volume to the appropriate number of cryopreservation straws.  Label and seal the straws per the manufacturer recommendations.

3. Place the straws horizontally at 4C for 30 minutes.

4. Place the cooled straws into a labeled goblet on a labeled aluminum cane, and hang the specimen over the liquid level of the nitrogen, well below the frost line of the tank, for 30 minutes.

5. Plunge the specimens into liquid nitrogen for continued storage.

Note: a small test specimen may be cryopreserved, then thawed to determine success of the cryopreservation procedure.

 

Specimen thawing and processing:

On the day of IVF, the specimens are thawed for 10 minutes in a 37C water bath, and processed according to the procedure required, e.g. gradient preparation, swim-up, swim-out.

The method used in this laboratory for freezing intact testicular tissue is as follows, adapted from Alan and Cotman, 1997, is as follows:

Equipment:

Cryopreservation straws; 0.5ml (cryo vials may also be used)

Sterile pipettes, as needed

Cryopreservation medium (commercial) with glycerol as the cryoprotectant with egg yolk

Freezer (approximately -28 to 30C) with shelf space for horizontal aluminum cryo canes

Liquid nitrogen tank; room to hang specimen below the frost line of tank, and above liquid level

1. Testicular tubules are rinsed in hepes-buffered medium to eliminate or reduce RBC contamination.  The tubules are sectioned with a scalpel blade into pieces approximately 2-3mm in size, and placed into a 35mm dish that contains room temperature hepes-buffered medium and cryoprotectant medium (Freezing Medium – TYB with glycerol and gentamicin; Irvine Scientific, Irvine CA), mixed gently to 1:1 v/v.  Start a timer for 30 minutes.

2. Load the tissue pieces into as many cryo straws as required.  The 30 minute room temperature equilibration period will be ongoing during the loading process.  Seal and label the straws per the manufacturer recommendations.

3. At the end of the 30 minute room temperature equilibration, load the straws into as many goblets and onto as many aluminum cryo canes as needed.  Place the cryo canes with the specimen straws horizontally into the freezer for 30 minutes.

4. At the end of the 30 minute cooling period, hang the specimens over the liquid level of the nitrogen, well below the frost line of the tank, for 30 minutes.

5. Plunge the specimens into liquid nitrogen for continued storage.

Note: a small test specimen may be cryopreserved, then thawed to determine success of the cryopreservation procedure.

Specimen thawing and processing for ICSI:

On the day of IVF, the straws with the intact tubules are thawed in a room temperature water bath for 10 minutes.  The contents are then expelled into room temperature hepes-buffered medium, rinsed into fresh hepes-buffered medium, and allowed to stand for 10 minutes prior to processing.  The tissue is placed on a sterile plastic petri dish with a small volume of hepes-buffered medium (enough to cover the tissue) and homogenized using sterile 18g needles and a #10 scalpel.  A small volume of the same medium is added to the homogenate, which is moved, without large tissue pieces, to a hepes-buffered medium drop under oil.  The homogenate is allowed to settle for at least 15 minutes before use.  Motility is evaluated in the drop prior to ICSI.  If needed, a 10mM stock solution of pentoxifylline is prepared to induce motility.

Using a pulled pipette, a small volume of the homogenate is moved to the ICSI dish, and motile sperm are captured using an assisted hatching pipette.  The captured sperm are moved to predetermined site in a drop of PVP (e.g. upper edge), and at the time the sperm are deposited the tail is struck with the assisted hatching pipette.  If pentoxifylline is required to induce motility, a small volume of homogenate is placed with an equal and small volume of the 10mM pentoxifylline stock solution.  This volume is moved to the ICSI dish as described earlier, for selection and movement of sperm to the PVP drop.

Note that overnight incubation of the cryopreserved, thawed, and processed homogenate may increase the effective yield of the preparation (Schiewe et al, 2004).

References

Allan JA, Cotman AS.  A new method for freezing testicular biopsy sperm: three pregnancies with sperm extracted from cryopreserved sections of seminiferous tubule.  Fertil Steril 1997;68:741-744.

Ben-Yosef D, Yogev L, Hauser R, Yavetz H, Azem F, Yovel I, Lessing JB, Amit A. Testicular sperm retrieval and cryopreservation prior to initiating ovarian stimulation as the first line approach in patients with non-obstructive azoospermia.  Hum Reprod 1999;14:1794-1801.

Chan PT, Schlegel PN.  Diagnostic and therapeutic testis biopsy.  Curr Urol Rep 2000;1:266-272.

Crabbé E, Verheyen G, Silber S, Tournaye H, Van de Velde H, Goossens A, Van Steirteghem A. Enzymatic digestion of testicular tissue may rescue the intracytoplasmic sperm injection cycle in some patients with non-obstructive azoospermia.  Hum Reprod 1998;13:2791-2796.

Friedler S, Raziel A, Soffer Y, Strassburger D, Komarovsky D, Ron-el R. Intracytoplasmic injection of fresh and cryopreserved testicular spermatozoa in patients with nonobstructive azoospermia--a comparative study.  Fertil Steril 1997;68:892-897.

Gil-Salom M, Romero J, Minguez Y, Rubio C, De los Santos MJ, Remohí J, Pellicer A. Pregnancies after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa.  Hum Reprod 1996;11:1309-1313.

Giorgetti C, Chinchole JM, Hans E, Charles O, Franquebalme JP, Glowaczower E, Salzmann J, Terriou P, Roulier R. Crude cumulative delivery rate following ICSI using intentionally frozen-thawed testicular spermatozoa in 51 men with non-obstructive azoospermia. Reprod Biomed Online 2005;11:319-324.

Griveau JF, Lobel B, Laurent MC, Michardière L, Le Lannou D. Interest of pentoxifylline in ICSI with frozen-thawed testicular spermatozoa from patients with non-obstructive azoospermia.  Reprod Biomed Online 2006;12:14-18.

Huang FJ, Chang SY, Tsai MY, Kung FT, Lin YC, Wu JF, Lu YJ.  Clinical implications of intracytoplasmic sperm injection using cryopreserved testicular spermatozoa from men with azoospermia. J Reprod Med 2000;45:310-316.

Johnson L, Neaves WB, Barnard JJ, Keillor GE, Brown SW, Yanagimachi R.  A comparative morphological study of human germ cells in vitro or in situ within seminiferous tubules.  Biol Reprod 1999;61:927-934.

Johnson L, Staub C, Neaves WB, Yanagimachi R.  Live human germ cells in the context of their spermatogenic stages.  Hum Reprod 2001;16:1575-1582.

Kovacic B, Vlaisavljevic V, Reljic M. Clinical use of pentoxifylline for activation of immotile testicular sperm before ICSI in patients with azoospermia.  J Androl2006;27:45-52.

Round spermatid nucleus injection (ROSNI).  The Practice Committee of the American Society for Reproductive Medicine and the Practice Committee of the Society for Assisted Reproductive Technology.  Fertil Steril 2008;90:S199-S201.

Schiewe MC, Ringler GE, Bennett CJ, Rothman CM, Mars RP.  Cryopreservation of intact testis biopsies for ICSI.  Fertil Steril 1997;68 Suppl 1:S115.

Schiewe M, Hubert G, Buyalos R.  Testicular tissue processing: maximizing viability for cryopreservation and clinical use.  Fertil Steril 2004;81 Suppl 3:15.

Schlegel PN. Testicular sperm extraction: microdissection improves yield with minimal tissue excision.  Hum Reprod 1999;14:131-135.

Silber SJ.  Microsurgical TESE and the distribution of spermatogenesis in non-obstructive azoospermia.  Hum Reprod. 2000;5:2278-2284.

Silber SJ, Nagy Z, Devroey P, Tournaye H, Van Steirteghem AC.  Distribution of spermatogenesis in the testicles of azoospermic men: the presence or absence of spermatids in the testes of men with germinal failure.  Hum Reprod 1997;12:2422-2428.

Taşdemir I, Taşdemir M, Tavukçuoğlu S. Effect of pentoxifylline on immotile testicular spermatozoa.  J Assist Reprod Genet 1998;15:90-92.

Terriou P, Hans E, Giorgetti C, Spach JL, Salzmann J, Urrutia V, Roulier R.  Pentoxifylline initiates motility in spontaneously immotile epididymal and testicular spermatozoa and allows normal fertilization, pregnancy, and birth after intracytoplasmic sperm injection.  J Assist Reprod Genet 2000;17:194-199.

Verheyen G, Vernaeve V, Van Landuyt L, Tournaye H, Devroey P, Van Steirteghem A.  Should diagnostic testicular sperm retrieval followed by cryopreservation for later ICSI be the procedure of choice for all patients with non-obstructive azoospermia?  Hum Reprod 2004;19:2822-2830.