Clinical use of spermatid ค Introduction ค Spermiogenesis block ค Prognostic factors of spermatid conception ค Potential health hazards ค Practical recommendation
Spermatogenesis Spermatogonia 46XY Mitosis 1ฐ Spermatocyte 46XY46XY Meiosis I 2ฐ Spermatocyte 23X23Y Meiosis II Spermatid 23X 23X 23Y 23Y Spermiogenesis Spermatozoa 23X,23Y
ค Spermiogenesis failure : 1-2% of all infertility ค Ogura et al 1993 - Fertilization of hamster oocyte from ROSI ค Full term delivery of mouse and rabbit oocyte from spermatid injection (Ogura 1994,Kimura 1995,Sofikitis 1994) ค First fertilization of human oocyte from spermatids in 1995 by Vanderzwalmen P.,et al ค First birth of two infants from spermatid conception in 1995 by Tesazik J. , et al.
Outcome of spermatids injection (ROSI and ELSI) ROSI ELSI ROSI ELSI ROSI* ELSI* ROSI ELSI ROSI ELSI ELSI Preg. 1 FT 2 FT 1 OP 1 MC 2 OP 2 OP 1 MC Authors Vanderzwalmen P Fishel S, Antinori S, Tesarik J, Sofikitis NU, * Ejaculated spermatids FR 22% 64% 22% 38% 33% 18% 45% 44% 76.5%
Spermatid conceptions ค 3 babies from ROSI ค 9 babies from ELSI คHighly variable and unpredictable outcomes of spermatid conception ค All nuclear and cytoplasmic modification may not fully development ค Possible health hazards
Spermatid classification ค The round S. (Sa, Sb1) ค The elongating S. (Sb2) ค The elongated S. (Sc,Sd1) ค The late elongated S.(Sd2) or Mature spermatids
Spermiogenesis block ค Aetiologic factors ค Genetic factors (Mouse model) - White spotting locus (encoding c-kit receptor) - Sl locus (encoding c-kit ligand) - Retinoic acid receptor a gene - Retinoic X receptor b gene
คNon-genetic factors - Radio and chemotherapy - Long-term estrogen therapy - Nutrition - Heat - Infections - Endocrinopathies - Cryptorchidism - Idiopathic
คEjaculated spermatids from spermiogenesis arrest patients - Not related to serum FSH - Abnormal relationship between spermatids and Sertoli cells t Priming Sertoli cells with FSH
คSpermiogenesis failure - Complete : < round spermatids - Incomplete : > elongated spermatids.
คRound spermatids in ejaculate and testicular biopsy from patients undergoing a spermatid conception attempt. - 1/2 - Round spermatid - 1/2 - Elongated spermatid or no spematid
66 patients with complete spermiogenesis failure Biopsy + - + Ejaculate + + - No (%) of patients 30(45) 27(41) 9(14) (Tesarik J., et al. Human Reproduction 1998 ;13 Suppl 3 : 89-107)
- A few foci produced ROS - ROS production has a cyclic character - Prematurely released spermatocytes develop into spermatids - Spermatids from biopsy have a higher biologic quality, fertilizing ability
Prognostic factors of spermatid conception คComplete and incomplete spermiogenesis failure ค Genetic anomalies ค Epigenic disorders ค Oocyte-activating factor
Complete and incomplete spermiogenesis failure. Amer M.,etal (Lancet 1997;350 (9071) : 116) ค59 cases ค 2 Ongoing clinical pregnancies - from 3 ELSI ค 4 Biochemical pregnancies - from ROSI ค Incomplete spermiogenesis failure is better
ค Fertilization and cleavage rates in complete spermiogenesis failure ค Factors at implantation and post-implantation period.
Genetic anomalies. คHuman spermatogenic disorder - YRRM 1 , YRRM 2 , DA2 genes : at long arm Y, AZF region - DAZLA : at chromosome 3 - Polygenic control ค Affecting sperm differentiation and function ค Not affecting fertilization, embryonic and fetal development
Epigenic disorders. คNuclear protein ค Centriole
Nuclear protein ค Nuclear condensation:spermatid spermatozoa - Histones, Protamines ค Nuclear decondensation:After fertilization - Oocyte cytoplasmic factor : S-S bond reduction - Histones, Protamines
ค May alter the normal sequence of early post-fertilization chromatin functions ค Rapid appearance of PN after ROSI and ROSNI (<10 hours) ค Abnormal pronuclear development - Persistence of a very small dense nucleus - Prolong appearance of a single syngamic nucleus
Centriole คSpermatids : - Proximal and distal centrioles - Distal centriole disappears in late elongated spermatids. คMicrotubule-organizing center(MTOC) - Zygote microtubules - Mitotic spindle
ค Mature sperm cell : 1 centriole associated with MTOC ค Distal centriole of spermatid - May associate with MTOC - Multipolar mitotic spindle - Chromosomal abnormalities
Oocyte activating factor ค Sperm-induced activation of human oocytes. - Ca2+ spiking or oscillations - Dependent on the oocyte-activating factor in cytoplasm of sperm - Oscillin
คSousa M.,et al. (Mol. Hum. Reprod. 1996; 2(11) : 853-7) - Ca2+ oscillation occurs in ROSI but not after the injection of 1ฐ or 2ฐ spermatocyte or PMN - Spermatids from men who produced normal mature sperm
Oocyte activating factor (Cont.) ค Tesarik J., et al. (Hum Repecd 1998; B supple 3:89-107) - Spermatids from men with complete spermiogenesis failure - 6 in 36 oocytes (17%) showed Ca2+ oscillation at 30-60 min - 2 in 36 oocytes (6%) showed Ca2+oscillation at 2-3hr.
Oocyte activating factor (Cont.) ค Oocyte activating factor may be deficient ค Fertilization , implanlation, abortion ค Quality of Ca2+ signal in fertilized oocytes influences developmental process.
Potential health hazards ค Transmission of infertility genes คGenomic 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 recommendation ค Preparation of testicular suspension ค Identification of spermatids ค Quality assurance ค Medical counseling
Preparation of testicular cell suspension ค Tissue dissociation - Mechanical : 2 glass slides or fine needle - Enzymatic digestion : collagenase type IV Erythrocyte lysing buffer treatment ค Separation of different cell - Base on cell size or density - Centrifugal elutriation technique ( Blanchard et al. 1991)
Identification of round spermatids ค Diameter : 6.5-8 mm, equal to RBC and small lymphocyte ค Clearly visible nucleus. ค Acrosomal structure : bright spot , small protusion - not observed in golgi phase spermatid ค 3 dimensionally round cell - Sertoli cell nucleus : flat, transparent, prominent central nucleolus
Quality of spermatids ค Viability ค Genomically abnormality - No reliable method except staining or destroying the cells. - No lysis after aspiration in micropipette - Spermatid in vitro culture
Quality assurance ค Recognition of living round spermatids - Skill ค Injection technique ค Timing of PN development - More frequent inspection ค Frequent development of a large syngamy nucleus
ค Spermatid and spermatocyte cryopreservation - ROSI from frozen-thawed specimen pregnancy - Avoid the need for a new surgical intervention - Progression from maturation arrest to Sertoli-cell only syndrome - Preservation the individual’s reproductive potential.
Medical counseling ค Take-home-baby rate 5-10% in incomplete spermiogenesis failure ค Take-home-baby rate 1-5% in - Complete spermiogenesis failure - Wife’s age approaches 40 years. ค Potential health hazard ค Success rate are likely to improve in the near future