Clinical use of spermatidClinical use of spermatid

คค IntroductionIntroduction

คค Spermiogenesis blockSpermiogenesis block

คค Prognostic factors of spermatid conceptionPrognostic factors of spermatid conception

คค Potential health hazardsPotential health hazards

คค Practical recommendationPractical recommendation

SpermatogenesisSpermatogenesis

SpermatogoniaSpermatogonia 46XY46XYMitosisMitosis

11คค SpermatocyteSpermatocyte 46XY 46XY 46XY46XYMeiosis IMeiosis I

22คค SpermatocyteSpermatocyte 23X 23X 23Y23YMeiosis IIMeiosis II

SpermatidSpermatid 23X 23X23X 23X 23Y 23Y 23Y 23Y

SpermiogenesisSpermiogenesis

SpermatozoaSpermatozoa 23X,23Y23X,23Y

คค Spermiogenesis failure : 1-2% of all Spermiogenesis failure : 1-2% of all

infertilityinfertility

คค Ogura et al 1993Ogura et al 1993

- F- Ferertilization of hamster oocyte from ROSI tilization of hamster oocyte from ROSI

คค Full term delivery of mouse and rabbit oocyte Full term delivery of mouse and rabbit oocyte

from spermatid injection (Ogura 1994,Kimura from spermatid injection (Ogura 1994,Kimura

1995,Sofikitis 1994)1995,Sofikitis 1994)

คค First fertilization of human oocyte from First fertilization of human oocyte from

spermatids in 1995 by Vanderzwalmen P.,et alspermatids in 1995 by Vanderzwalmen P.,et al

คค First birth of two infants from spermatid First birth of two infants from spermatid

conception in 1995 by Tesazik J. , et al.conception in 1995 by Tesazik J. , et al.

Outcome of spermatids injection (ROSI and ELSI)Outcome of spermatids injection (ROSI and ELSI) AuthorsAuthors

Vanderzwalmen PVanderzwalmen P

Fishel S,Fishel S,

Antinori S,Antinori S,

Tesarik J,Tesarik J,

Sofikitis NU,Sofikitis NU,

* Ejaculated spermatids* Ejaculated spermatids

ROSIROSIELSIELSI

ROSIROSIELSIELSIROSI*ROSI*ELSI*ELSI*ROSIROSIELSIELSI

ROSIROSIELSIELSIELSIELSI

FRFR

22%22%64%64%

22%22%38%38%33%33%18%18%

45%45%44%44%76.5%76.5%

Preg.Preg.

1 FT1 FT2 FT2 FT1 OP1 OP1 MC1 MC

2 OP2 OP2 OP2 OP1 MC1 MC

คค 3 babies from ROSI3 babies from ROSIคค 9 babies from ELSI9 babies from ELSI

Spermatid conceptionsSpermatid conceptions

คค Highly variable and unpredictable Highly variable and unpredictable outcomes of spermatid conceptionoutcomes of spermatid conceptionคค All nuclear and cytoplasmic All nuclear and cytoplasmic modification may not fully modification may not fully development development คค Possible health hazards Possible health hazards

Spermatid classificationSpermatid classification

คค The round S. (Sa, Sb1)The round S. (Sa, Sb1)

คค The elongating S. (Sb2)The elongating S. (Sb2)

คค The elongated S. (Sc,Sd1)The elongated S. (Sc,Sd1)

คค The late elongated S.(Sd2) or The late elongated S.(Sd2) or

Mature spermatidsMature spermatids

Spermiogenesis blockSpermiogenesis block

คค Aetiologic factorsAetiologic factors

คคคค คค Genetic factors (Mouse model)Genetic factors (Mouse model)

คคคคคค-- White spotting locus (encoding White spotting locus (encoding

c-kit receptor)c-kit receptor)

คคคคคค-- Sl locus (encoding c-kit ligand) Sl locus (encoding c-kit ligand)

คคคคคค-- Retinoic acid receptor Retinoic acid receptor genegene

- Retinoic X receptor - Retinoic X receptor gene gene

คคคค Non-genetic factorsNon-genetic factorsคคคค- Radio and chemotherapy- Radio and chemotherapy

- Long-term estrogen therapy- Long-term estrogen therapy- Nutrition- Nutrition- Heat- Heat- Infections- Infections- Endocrinopathies- Endocrinopathies- Cryptorchidism- Cryptorchidism- Idiopathic- Idiopathic

คค Ejaculated spermatids from Ejaculated spermatids from spermiogenesis arrest patientsspermiogenesis arrest patientsคค- Not related to serum FSH- Not related to serum FSH

- Abnormal relationship - Abnormal relationship between spermatids and between spermatids and Sertoli cellsSertoli cells Priming Sertoli cells with FSHPriming Sertoli cells with FSH

คค Spermiogenesis failureSpermiogenesis failureคค- Complete : - Complete : << round spermatids round spermatids

- Incomplete : - Incomplete : >> elongated elongated spermatids.spermatids.

คค Round spermatids in ejaculate and Round spermatids in ejaculate and testicular biopsy from patients testicular biopsy from patients undergoing a spermatid conception undergoing a spermatid conception attempt. attempt.

- 1/2 - Round spermatid- 1/2 - Round spermatid- 1/2 - Elongated spermatid or no - 1/2 - Elongated spermatid or no

spematidspematid

EjaculateEjaculate

++++--

BiopsyBiopsy

++--++

No (%) of patientsNo (%) of patients

30(45)30(45)27(41)27(41)9(14)9(14)

(Tesarik J., et al. Human Reproduction 1998 ;13 (Tesarik J., et al. Human Reproduction 1998 ;13 Suppl 3 : 89-107)Suppl 3 : 89-107)

66 patients with complete spermiogenesis failure66 patients with complete spermiogenesis failure

- A - A few foci produced ROSfew foci produced ROS- ROS production has a cyclic - ROS production has a cyclic character character - Prematurely released - Prematurely released spermatocytes develop into spermatocytes develop into spermatids spermatids - Spermatids from biopsy have a - Spermatids from biopsy have a higher biologic quality, fertilizing higher biologic quality, fertilizing abilityability

Prognostic factors of spermatid Prognostic factors of spermatid conceptionconception

คค Complete and incomplete spermiogenesis Complete and incomplete spermiogenesis

failurefailure

คค Genetic anomaliesGenetic anomalies

คค Epigenic disordersEpigenic disorders

คค Oocyte-activating factorOocyte-activating factor

Complete and incomplete Complete and incomplete spermiogenesis failure.spermiogenesis failure.

Amer M.,etal (Lancet 1997;350 (9071) : 116)Amer M.,etal (Lancet 1997;350 (9071) : 116)คค 59 cases59 cases

คค 2 Ongoing clinical pregnancies - from 3 2 Ongoing clinical pregnancies - from 3

ELSIELSI

คค 4 Biochemical pregnancies - from ROSI4 Biochemical pregnancies - from ROSI

คค Incomplete spermiogenesis failure is Incomplete spermiogenesis failure is

better better

คค Fertilization and cleavage rates in Fertilization and cleavage rates in complete spermiogenesis failurecomplete spermiogenesis failureคค Factors at implantation and Factors at implantation and post-implantation period.post-implantation period.

Genetic anomalies.Genetic anomalies.คค Human spermatogenic disorderHuman spermatogenic disorder

คคคค- YRRM 1 , YRRM 2 , DA2 genes : at - YRRM 1 , YRRM 2 , DA2 genes : at

long arm Y, AZF regionlong arm Y, AZF region

- DAZLA : at chromosome 3- DAZLA : at chromosome 3

- Polygenic control- Polygenic control

คค Affecting sperm differentiation and functionAffecting sperm differentiation and function

คค Not affecting fertilization, embryonic and Not affecting fertilization, embryonic and

fetal developmentfetal development

Epigenic disorders.Epigenic disorders.

คค Nuclear proteinNuclear protein

คค CentrioleCentriole

Nuclear proteinNuclear protein

คค Nuclear condensation:spermatid spermatozoaNuclear condensation:spermatid spermatozoa

คคคค- Histones, Protamines- Histones, Protamines

คค Nuclear decondensation:After fertilizationNuclear decondensation:After fertilization

- Oocyte cytoplasmic factor : S-S bond - Oocyte cytoplasmic factor : S-S bond

reductionreduction

- Histones, Protamines- Histones, Protamines

คค May alter the normal sequence of early May alter the normal sequence of early

post-fertilization chromatin functionspost-fertilization chromatin functions

คค Rapid appearance of PN after ROSI and ROSNI Rapid appearance of PN after ROSI and ROSNI

(<10 hours)(<10 hours)

คค Abnormal pronuclear developmentAbnormal pronuclear development

- Persistence of a very small dense nucleus - Persistence of a very small dense nucleus

- Prolong appearance of a single syngamic - Prolong appearance of a single syngamic

nucleus nucleus

CentrioleCentrioleคค Spermatids : Spermatids :

- Proximal and distal centrioles- Proximal and distal centrioles

- Distal centriole disappears in late - Distal centriole disappears in late

elongated spermatids.elongated spermatids.

คค Microtubule-organizing center(MTOC)Microtubule-organizing center(MTOC)

- Zygote microtubules- Zygote microtubules

- Mitotic spindle- Mitotic spindle

คค Mature sperm cell : 1 centriole Mature sperm cell : 1 centriole associated with MTOCassociated with MTOCคค Distal centriole of spermatidDistal centriole of spermatid

- May associate with MTOC- May associate with MTOC- Multipolar mitotic spindle- Multipolar mitotic spindle- Chromosomal abnormalities- Chromosomal abnormalities

Oocyte activating factorOocyte activating factor

คค Sperm-induced activation of human Sperm-induced activation of human

oocytes. oocytes.

- Ca- Ca2+2+ spiking or oscillations spiking or oscillations

- Dependent on the oocyte-activating - Dependent on the oocyte-activating

factor in cytoplasm of sperm factor in cytoplasm of sperm

- Oscillin- Oscillin

คค Sousa M.,et al. (Mol. Hum. Reprod. Sousa M.,et al. (Mol. Hum. Reprod. 1996; 2(11) : 853-7)1996; 2(11) : 853-7)

- Ca2+ oscillation occurs in ROSI but - Ca2+ oscillation occurs in ROSI but not after the injection of 1not after the injection of 1คคor 2or 2คคspermatocyte or PMN spermatocyte or PMN

- Spermatids from men who produced - Spermatids from men who produced normal mature sperm normal mature sperm

Oocyte activating factor (Cont.)Oocyte activating factor (Cont.)คค Tesarik J., et al. (Hum Repecd 1998; B Tesarik J., et al. (Hum Repecd 1998; B

supple 3:89-107)supple 3:89-107)

- Spermatids from men with complete - Spermatids from men with complete

spermiogenesis failurespermiogenesis failure

- 6 in 36 oocytes (17%) showed Ca- 6 in 36 oocytes (17%) showed Ca2+2+ oscillation at 30-60 min oscillation at 30-60 min

- 2 in 36 oocytes (6%) showed - 2 in 36 oocytes (6%) showed

CaCa2+2+oscillation at 2-3hr.oscillation at 2-3hr.

Oocyte activating factor (Cont.)Oocyte activating factor (Cont.)

คค Oocyte activating factor may be deficientOocyte activating factor may be deficient

คค Fertilization , implanlation, abortionFertilization , implanlation, abortion

คค Quality of CaQuality of Ca2+2+ signal in fertilized signal in fertilized

oocytes influences developmental process.oocytes influences developmental process.

Potential health hazardsPotential health hazardsคค Transmission of infertility genes

คค Genomic imprinting abnormalities

- Uncertainty of whether the process is

actually completed

- IGF2,p57KIP2,H19,SNRPN,PEG1/MEST

- Wilms’tumor, Adrenocortical CA.,

Rhabdomyosarcoma,

Beckwith-Wiedemann syndrome, Prader-Willi

syndome

- No available method to detect

Practical recommendationPractical recommendation

คค Preparation of testicular suspensionPreparation of testicular suspension

คค Identification of spermatidsIdentification of spermatids

คค Quality assuranceQuality assurance

คค Medical counselingMedical counseling

Preparation of testicular cell suspensionPreparation of testicular cell suspension

คค Tissue dissociationTissue dissociation

- Mechanical : 2 glass slides or fine - Mechanical : 2 glass slides or fine

needleneedle

- Enzymatic digestion : collagenase - Enzymatic digestion : collagenase

type IV type IV

Erythrocyte lysing buffer treatmentErythrocyte lysing buffer treatment

คค Separation of different cellSeparation of different cell

- Base on cell size or density- Base on cell size or density

- Centrifugal elutriation technique- Centrifugal elutriation technique

( Blanchard et al. 1991)( Blanchard et al. 1991)

Identification of round spermatidsIdentification of round spermatidsคค Diameter : 6.5-8Diameter : 6.5-8m, equal to RBC and m, equal to RBC and

small lymphocytesmall lymphocyte

คค Clearly visible nucleus.Clearly visible nucleus.

คค Acrosomal structure : bright spot , small Acrosomal structure : bright spot , small

protusionprotusion

- not observed in golgi phase spermatid- not observed in golgi phase spermatid

คค 3 dimensionally round cell3 dimensionally round cell

- Sertoli cell nucleus : flat, transparent, - Sertoli cell nucleus : flat, transparent,

prominent central nucleolusprominent central nucleolus

Quality of spermatidsQuality of spermatidsคค ViabilityViability

คค Genomically abnormalityGenomically abnormality

- No reliable method except staining or - No reliable method except staining or

destroying the cells.destroying the cells.

- No lysis after aspiration in micropipette- No lysis after aspiration in micropipette

- Spermatid in vitro culture- Spermatid in vitro culture

Quality assuranceQuality assuranceคค Recognition of living round spermatidsRecognition of living round spermatids

- Skill- Skill

คค Injection techniqueInjection technique

คค Timing of PN developmentTiming of PN development

- More frequent inspection- More frequent inspection

คค Frequent development of a large Frequent development of a large

syngamy nucleussyngamy nucleus

คค Spermatid and spermatocyte Spermatid and spermatocyte

cryopreservationcryopreservation

- ROSI from frozen-thawed specimen - ROSI from frozen-thawed specimen

pregnancypregnancy

- Avoid the need for a new surgical - Avoid the need for a new surgical

interventionintervention

- Progression from maturation arrest - Progression from maturation arrest

to Sertoli-cell only syndrometo Sertoli-cell only syndrome

- Preservation the individual’s - Preservation the individual’s

reproductive potential.reproductive potential.

Medical counselingMedical counselingคค Take-home-baby rate 5-10% in incomplete Take-home-baby rate 5-10% in incomplete

spermiogenesis failurespermiogenesis failure

คค Take-home-baby rate 1-5% inTake-home-baby rate 1-5% in

- Complete spermiogenesis failure- Complete spermiogenesis failure

- Wife’s age approaches 40 years.- Wife’s age approaches 40 years.

คค Potential health hazardPotential health hazard

คค Success rate are likely to improve in the Success rate are likely to improve in the

near future near future