P-059 Sperm DNA fragmentation (SDF) after cryopreservation and sperm selection: implications for clinical pregnancies and live births after intrauterine insemination with donor sperm
Authors: Chimienti T , Punjabi U , Goovaerts I , Peeters K , Bouziotis J , De Neubourg D 
Published: June 2023

Abstract

Study question
Can SDF assessment pre-cryopreservation, post-thaw and after post-thaw sperm selection predict clinical outcome in a heterologous intra-uterine donor insemination program (IUI)?

Summary answer
SDF increased post-cryopreservation in donor sperm and after post-thaw density gradient without affecting clinical pregnancy, live birth and miscarriage rates.

What is known already
Sperm cryopreservation is effective for preservation of male fertility and facilitates the storage of donor semen, while infectious disease screening can be completed and confirmed negative. Sperm viability and motility are the most vulnerable parameters during the freeze-thaw process. Inherent reactive oxygen species may induce SDF via oxidative stress. Controversial results are reported regarding induction of SDF after cryopreservation due to differences in cryo tolerance, methods of cryopreservation and methods used to evaluate SDF. Sperm selection post-thaw is indispensable to select a normal motile fraction for insemination. Again, results are controversial regarding influence on SDF levels after sperm selection post-thaw.

Study design, size, duration
This was a prospective, observational study. Between March 2015 and March 2019, eighteen potential sperm donors were screened via a three step plan – semen assessment, medical assessment and serological blood tests. In accordance with the Belgian legislation (2007), a sperm donor was matched to obtain a maximum of 6 pregnancies in acceptor women/couples, allowing for more than one offspring/woman or couple. A total of 106 acceptors were matched for heterologous intra-uterine insemination.

Participants/materials, setting, methods
Semen samples were collected after 2-7 days abstinence and standard semen parameters analyzed within 60 mins after ejaculation. Within the same time frame SDF test was performed using TUNEL assay both before and after cryopreservation and after a two-step discontinuous density gradient centrifugation post-thaw. Samples were cryopreserved by the conventional slow freezing method using sperm freeze solution. A yield of ≥ 2 M progressive spermatozoa post-thaw after selection was sufficient to contemplate IUI.

Main results and the role of chance
Mean female age at first cycle was 33.9 ± 4.1 years and donor age 28.5 ± 5.6 years. Out of 429 cycles, 100 (23.3%) resulted in clinical pregnancy. Excluding three ongoing pregnancies, we counted 75 live births (17.6% of cycles or 77.3% of pregnancies), while 21 pregnancies ended in miscarriage (4.9% of cycles or 21.6% of pregnancies) and 1 resulted in stillbirth.
Progressive motility (59.3 ± 12.5%) decreased significantly after cryopreservation (30.0 ± 13.2%; p &lt; 0.001), but increased post-thaw after density gradient centrifugation (61.6 ± 16.0%; p &lt; 0.001). On the contrary, SDF (12.0 ± 5.9%) increased post-cryopreservation (26.3 ± 14.5%; p &lt; 0.001) and further increased after sperm selection (34.9 ± 22.1%; p = 0.04).
Using multilevel mixed-effects logistic regression (Odds ratio [95%CI]), we found that female age significantly influenced clinical pregnancy (0.911 [0.847-0.981]; p = 0.01), live birth (0.894 [0.834-0.959]; p = 0.002) and miscarriage rates (1.180 [1.033-1.347]; p = 0.015). There was no significant age-adjusted effect of SDF post-thaw after density gradient selection on clinical pregnancy (1.007 [0.994-1.021]; p = 0.30), live birth (1.001 [0.988-1.014]; p = 0.84) or miscarriage (1.021 [0.997-1.046]; p = 0.08). Median time-to-live birth was 4 cycles. Analyses of time-to-live birth with mixed-effects Cox models revealed no significant association with progressive motility; SDF before or after cryopreservation and after sperm selection post-thaw.

Limitations, reasons for caution
Results concern a small donor population, with high normal semen parameters and should therefore be extrapolated cautiously to subfertile men with normal and subnormal semen parameters.

Wider implications of the findings
In a donor IUI program with strict selection on conventional sperm parameters, differences in SDF before and after cryopreservation and gradient centrifugation do not change IUI outcomes, although cryopreservation and centrifugation increase SDF. Findings could be useful for the optimization of sperm freezing and selection in different assisted reproductive procedures.

Trial registration number
not applicable