Novel Technologies for Selecting the Best Sperm for IVF and ICSI Denny Sakkas, Ph.D. Scientific Director, Boston IVF Waltham, MA, USA
Testing The Sperm Population NOW
Sperm DNA testing Although we are still far from establishing as strong a correlation in the human as seen in animals, the current data on sperm DNA testing would suggest the following:
1. The presence of a high fraction of spermatozoa showing DNA damage is a negative trait that reduces the chances to father a child. 2. A specific threshold or percentage of DNA-damaged sperm that is incompatible with pregnancy is not yet established. 3. The predictive power of the current sperm-dna integrity tests seem to lose their strength from natural conception to ICSI, passing through IUI and IVF.
The percentage of ejaculated human spermatozoa per sample containing DNA damaged nuclei in relation to sperm concentration >50 45 40 35 30 25 20 15 10 5 0 DNA damage <20% DNA damage >20% Sperm concentration 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41
Mean DFI Value Fig 1 The relationship between increasing Sperm DNA Fragmentation and Age (N=4,542) 26 24 22 20 DFI Age Groups Subset for alpha = 0.05 (years) N 1 2 3 4 <30 525 15.83 30 to <35 1387 16.33 35 to <40 1297 17.32 17.32 40 to <45 797 18.23 18.23 45 to <50 336 19.85 19.85 50 to <55 125 21.08 >55 75 24.23 P =.141.095.866 1.000 18 16 14 30< 30 to <35 35 to <40 40 to <45 45 to <50 50 to <55 55> Age Group (years) (Unpublished Data)
TUNEL positivity in ejaculated spermatozoa shows a negative correlation with blastocyst development [Seli et al. Fertil Steril 2004] 100 % Blastocysts 80 60 40 Spearman s Rho = - 0.4 P=0.004 20 0 0 20 40 60 80 100 % TUNEL Positive
TUNEL assay reactivity in Repeat Pregnancy Loss (RPL) 50 (21) % Postive TUNEL Reactivity 40 30 20 10 (42) (26) 0 RPL Patients General Population Fertile Donors (Carrell et al. Arch Androl 2003: 49, 49-55)
About 85% of unexplained RPL patients included in the study showed low values of sssdf and high values of dssdf
Paternal influences (Impact of sperm with nuclear DNA strand breaks) Animal Human Fertilization Embryo development Pregnancy Miscarriages Future generations?
Paternal influences become important in relation to treating male factor infertility Intracytoplasmic sperm injection (ICSI)
Selecting The Right Sperm NOW
Selection Methods to isolate the best sperm Density Surface Charge Morphological Characteristics Membrane Integrity Surgical
Abnormal Spermatogenesis Normal Spermatogenesis Spermatogonia Spermatocytes SC Early spermatids Elongated spermatids Mature spermatozoa ROS Eliminate Abnormal Sperm Electrophoretic Separation Microfluidics Annexin V MACS Selection of Best Sperm IMSI HA binding
Limiting harmful paternal influences Using density gradient centrifugation techniques to routinely prepare sperm samples and limit the % of DNA damaged sperm N Mean SEM Raw sample 140 10.9 0.5 t=13.6 90% PureSperm Fraction 140 3.79 0.3 P<0.0001
Novel selection methods for enhancing sperm quality Aitken has reported a novel electrophoretic sperm isolation technique that produces suspensions containing motile, viable, morphologically normal spermatozoa which exhibit lower levels of DNA damage (Ainsworth et al., 2005).
Schematic representations of the cartridge-based electrophoretic separation technology used for this study Ainsworth, C. et al. Hum. Reprod. 2005 20:2261-2270; doi:10.1093/humrep/dei024
Impact of electrophoretic treatment on DNA integrity measured as percentage of TUNEL-positive cells (mean {+/-} SEM) for residual and separated sperm populations (n=6) Ainsworth, C. et al. Hum. Reprod. 2005 20:2261-2270; doi:10.1093/humrep/dei024
Novel selection methods for enhancing sperm quality - IMSI Selection of sperm under high magnification prior to ICSI (Bartoov et al., 2001). X3670 X5880 X400
Intracytoplasmic Morphological Sperm Injection (IMSI): a prospective randomised trial [Antinori et al. RBM Online 16, 2008 p835] ICSI (n=219) IMSI (n=227) Mean age (years) 31.9 + 3.3 31.7 + 3.2 No. of Previous ICSI failures 1.45 + 1.57 1.46 + 1.69 No. of MII oocytes recovered 6.29 + 3.04 6.58 + 3.34 No. of injected oocytes 2.92 + 0.26 2.90 + 0.29 No. of 2PN 2.76 + 0.42 2.75 + 0.45 No. of transferred embryos / patient 2.37 + 0.67 2.47 + 0.68 Clinical Pregnancy Rate (%) 58/219 (26.5)* 89/227 (39.2) Implantation Rate (%) 59/521 (11.3)* ; 1 twin 97/560 (17.3) ; 8 twins Miscarriage Rate (%) 14/58 (24.1) 15/89 (16.9)
AUTHORS' CONCLUSIONS: Results from RCTs do not support the clinical use of IMSI. There is no evidence of effect on live birth or miscarriage and the evidence that IMSI improves clinical pregnancy is of very low quality. There is no indication that IMSI increases congenital abnormalities. Further trials are necessary to improve the evidence quality before recommending IMSI in clinical practice.
Novel selection methods for enhancing sperm quality Apoptotic Marker Proteins A technique using Magnetic cell sorting (MACS) using annexin V conjugated microbeads which eliminates apoptotic spermatozoa has also recently been reported (Said et al., 2005). Example of other markers Fas (Sakkas et al., 1993) p53 (Enciso et al., 2012) Bcl-x, Cleaved Parp etc.
Magnetic cell separation of Annexin V Positive sperm [Dirican et al. JARG 2008]
Zech-Selector (Seiringer et al, 2013 RBM Online)
Novel selection methods for enhancing sperm quality Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status (Huszar et al, Fertil. Steril. 2003)
Selection of normal spermatozoa prior to ICSI using petri dishes coated with HA a polysaccharide whose receptor is present on the membrane of good sperm Gabor Huszar Lab (Jakab et al., 2005)
Selection of sperm with low aneuploidy frequencies (X, Y and 17 disomy) using HA Gabor Huszar Lab 0.9 0.8 0.7 Oligozoospermic Patients (n=12) Normozoospermic Patients (n=12) 0.6 0.5 0.4 0.3 Washed sperm Isolate prep. HA bound. 0.2 0.1 0 X&Y 17 Diploidy X&Y 17 Diploidy (Jakab et al, 2005)
Hyaluronan selection of sperm prior to ICSI improved miscarriage rates
DNA damage is lower in testicular compared to ejaculated sperm Greco et al. (Hum Reprod, 2005) A. First study in which pregnancy rates using testicular vs. ejaculated sperm, with a pathological DFI, were compared B. Couples with repeated IVF failure (> 2 cycles) C. TESA-ICSI significantly improved clinical pregnancy rates in these couples 0% 45%
Testing The Sperm Population FUTURE
Developmental promoters in sperm lack DNA methylation, but acquire methylation during development. DNA methylation of the HOXD locus in the mature sperm (blue bars) or primary fibroblasts (orange line overlay)
The role of proteomics in understanding sperm cell biology. RJ Aitken and MA Baker. This fundamental information will create a basis for identifying key points in the posttesticular maturation of spermatozoa that might be targeted for contraceptive purposes or implicated in the defective sperm function observed in a significant proportion of infertile males.
Delivery of RNAs present in human sperm. [Krawetz, Nature Reviews, 2005, 6, p633] Platts et al. Human Molecular Genetics, 2007, 16, p763
Selecting The Right Sperm THE FUTURE
Raman spectra of human sperm cells from different classes: a, b Pear shaped cells c, d Small cells e, f Double cells g, h Normal sperm cells Highlighted areas indicate deficiencies in DNA packaging efficiency and relative protein content
Mallidis et al, HRU 2013
The presence of the Base Excision Repair Protein [PARP1] in human oocytes. Can the egg take care of poor sperm DNA?
Venn diagram of the responsibilities of Reproductive Failure: EGG 80% EMBRYO SPERM 10% UTERUS 10%
The Take Home Message Paternal effects on reproduction are evident and should be taken into account when a couple fails to obtain a live birth Selecting the best sperm has the potential to improve the chance of pregnancy and decrease the risk of miscarriage
The Take Home Message PERSONALIZED ART Get the Correct Egg SPERM Embryo and Uterus