JASON- Time to change sperm preparation methods- 3rd international RHG congress 19.10.18 finalJason Kasraie Consultant Embryologist
THE JOURNEY OF SPERM
• High vaginal deposition • Acidic pH • ‘Backflow’ • Immune response
1
1 2
• Peristaltic flow • Reduction in sperm numbers • Immune response
1 2
THE JOURNEY OF THE SPERM IN NATURAL CONCEPTION- FALLOPIAN TUBE
• Isthmic mucus ‘plug’ • ?Surface protein allows passage • Sperm epithelial ‘binding’
1 2
3 4
• Thermotaxis • Chemotaxis • 1 in 14,000,000?
1 2
SELECTION OF SPERM WITH SUPERIOR MOTILITY
NORMAL SPERM ABNORMAL SPERM
SELECTION OF SPERM WITH GREATER DNA INTEGRITY(?)
SELECTION OF SPERM WITH GREATER MEMBRANE INTEGRITY?
HOW ARE WE TRYING TO REPLICATE NATURE RIGHT NOW?
PROGRESS SO FAR- WIDELY ADOPTED IN CLINICAL PRACTICE
Sperm preparation technique
One stage ‘wash’ Edwards et al. 1969 ‘Swim-up’ Lopata et al. 1978 Two stage ‘wash’ Edwards et al. 1980 Density gradient Pertoft et al. 1978 Wash and ‘swim-up’ Mahadevan and Baker 1984 Migration sedimentation chamber Tea et al. 1984
DENSITY GRADIENT PREPARATION
Migration sedimentation chamber
Sperm preparation medium
SPERM WASH/ SWIM-UP/ MSC
WHAT ARE WE TRYING TO ACHIEVE IN- VITRO? ¢ Superior motility ¢ Superior morphology ¢ DNA integrity ¢ Remove seminal plasma ¢ Remove detritus ¢ Enable capacitation ¢ Inhibit premature acrosome reaction ¢ Minimise ROS ¢ Zero reprotoxicity ¢ Selection of a highly fecund sub-population of
sperm
Normal semen analysis = IUI/IVF
¢ Magnetic activated cell sorting (MACS) ¢ Hyaluronan binding assay ¢ Others
Author'(year) Sperm'selection'method (i)$Selection$based$on$surface$charge Ainsworth'et'al.'(2007) Electrophoretic'separation'(Microflow,'CS@10)
Vanderzwalmen'et'al.'(2008) IMSI
Mauri'et'al.'(2010) IMSI
Gianaroli'et'al.'(2008) ICSI'using'polarization'microscopy Gianaroli'et'al.'(2010) ICSI'using'polarization'microscopy
Tamer M. Said, Jolande A. Land; Effects of advanced selection methods on sperm quality and ART outcome: a systematic review, Human Reproduction Update, 2011, Vol 17:6 I
INTRACYTOPLASMIC MORPHOLOGICALLY SELECTED SPERM INJECTION (IMSI)
X400 (Hoffman) x12000 Nomarski differential Interference contrast
Vanderzwalmen et al, RBM online 2008
INTRACYTOPLASMIC MORPHOLOGICALLY SELECTED SPERM INJECTION (IMSI)
Bartoov et al, Journal of Andrology, 2002
EVIDENCE
¢ Sperm nuclei in subfertile men contained more intranuclear vacuoles than fertile controls1
¢ Sperm with vacuoles at ICSI = decreased PR & increased early miscarriage2
1. Mundi et al., 1994 2. Bartoov et al., 2001, 2002, 2003; Berkovitz et al., 2005, 2006; Franco et al., 2008; Antinori et al., 2008
MORPHOLOGICAL ASSESSMENT
Normal form, no vacuoles
Grade I 56.3
Normal form ≤2 vacuoles Grade II 61.4 Normal form ≥2 vacuoles or 1 large vacuole
Grade III 5.1
Grade IV 0.0
EVIDENCE FOR IMSI AS A TOOL FOR SPERM SELECTION
Figure 5. Forest plot of comparison: 1 Ultra high (IMSI) versus regular magnification (ICSI), outcome: 1.2 Clinical pregnancy per allocated couple.
EVIDENCE FOR IMSI AS A TOOL FOR SPERM SELECTION
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.
Live births
• Phospholipid phosphatidylserine (PS) is a marker of peri-apoptosis. • Bind to annexin V conjugated with magnetic beads
EVIDENCE FOR MACS AS A TOOL FOR SPERM SELECTION
J Assist Reprod Genet (2013) 30:479–485
¢ Paucity of high quality research after this but it may be safe:-
EVIDENCE FOR MACS AS A TOOL FOR SPERM SELECTION
Romany et al ‘Removal of annexin V–positive sperm cells for intracytoplasmic sperm injection in ovum donation cycles does not improve reproductive outcome: a controlled and randomized trial in unselected males’ Fertility and Sterility, Volume 102, Issue 6, 2014.
Romany L, Garrido N, Cobo A, Aparicio-Ruiz B, Serra V, Meseguer M. Obstetric and perinatal outcome of babies born from sperm selected by MACS from a randomized controlled trial. J Assist Reprod Genet 2017;342:201–7
HYALURONAN BINDING ASSAY/ SELECTION (PICSI)
Huszar et al, 2009
EVIDENCE FOR HYALURONAN BINDING AS A TOOL FOR SPERM SELECTION
Ronit Beck-Fruchter, Eliezer Shalev, Amir Weiss- Reproductive BioMedicine Online 2016 32, 286-298DOI: (10.1016/j.rbmo.2015.12.001)
Implantation rate
Clinical benefit using sperm hyaluronic acid binding technique in ICSI cycles: a systematic review and meta-analysis
EVIDENCE FOR HYALURONAN BINDING AS A TOOL FOR SELECTION
Age ≥ 35 Live Birth Miscarriage Odds Ratio (95% CI)
PICSI 182 29 0.50 ICSI 181 58 0.3 – 0.8
P = 0.0053
Significant reduction in miscarriage rate, but reduction confined to age ≥35
SOMETIMES SUGGESTED AS A TREATMENTS FOR…
¢ Recurrent implantation failure ¢ Severe male factor infertility ¢ Older couples ¢ Failed fertilisation at ICSI
OTHER TECHNIQUES
ELECTROPHORESIS/ ZETA POTENTIAL
Sperm with high negative surface electrical charge, named “Zeta potential”, are mature and more likely to have intact chromatin.
Ainsworth et al. 2005 Said & Land 2011
MICROFLUIDICS
Samuel et al Transl Androl Urol. 2018 Jul; 7(Suppl 3): S336-S347
MICROFLUIDICS FOR TESTICULAR SAMPLES?
Samuel et al Transl Androl Urol. 2018 Jul; 7(Suppl 3): S336-S347
AND FINALLY
WHAT SPERM CONCENTRATION DOES YOUR UNIT USE FOR OVERNIGHT INSEMINATION?
ACE survey
= Progressively motile sperm (millions/ml)
= Insemination volume in µl
CONCLUSIONS
¢ We probably don’t put as much effort into sperm preparation as we should
¢ Promising new techniques are on the horizon but there is a lack of high quality evidence for efficacy
¢ Further high quality research is required ¢ There is probably work to be undertaken to
improve and standardise the methods that we currently utilise
THANK YOU
Mytton Oak Rd Shrewsbury