Impact of DNA Fragmentation in Sperm on Pregnancy Outcome After Intra-uterine Insemination in a Spontaneous Cycle
Phase: NA
Conditions: Infertility Unexplained, Infertility, Male, DNA Damage
Sponsor: University Hospital, Antwerp

 
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Impact of DNA fragmentation in sperm on the pregnancy outcome after 
intra-uterine insemination in a spontaneous cycle 
 
NCT03319654 
 
Document date: September 7th 2017 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Introduction 
Infertility affects about 10% of all couples and is defined by a failure to achieve a clinical pregnancy 
within a year of regular unprotected sexual intercourse. (Boivin 2007, Zegers-Hochschild 2009 ). 
Male infertility accounts for nearly 30% of diagnoses, whilst an estimated 40% of male factor 
infertility remains unexplained (de Kretser 1997) and most of male factor infertility cannot be treated 
adequately.  
The current diagnosis of male infertility is based on semen criteria updated by the WHO in 2010. These 
guidelines would consider a man normal with reference values of greater than 15 million sperm, greater 
than 4% normal morphology, and 32% progressive motility. By doing so more men are considered 
fertile while there may be an unnoticed rise in the number of infertile men meantime being assigned to 
the ‘unexplained’ category. Furthermore , although the WHO classification suggests accuracy through 
a methodology harmonized across laboratories (Wang 2014), the relevance for the choice of treatment 
and the predictive value for an infertile couple embarking on medically assisted reproduction 
technologies (MAR) is questionable. (Hamilton 2015)  
In other words, there is a strong clinical need to distinguish fertile from infertile men through new 
sperm function testing and to be able to select both the patient population who will benefit from MAR 
as well as the type of treatment. 
Numerous studies utilizing different techniques for assessing sperm DNA fragmentation support the 
existence of a significant association between sperm DNA damage and pregnancy outcomes (Collins et 
al., 2008). Moreover, a significant number of subfertile men have abnormal DNA integrity despite 
normal semen parameters (Kodama et al., 1997; Spano et al., 2000; Zini et al., 2001). The level of 
DNA fragmentation correlates negatively with pregnancy and delivery in both natural and assisted 
conceptions (Evenson & Wixon 2006; Zhang et al. 2008; Zini 2011).  
Our laboratory has set up a program where direct DNA fragmentation testing with terminal 
deoxyuridine nick end labeling (TUNEL) assay (Mitchell 2011) on fresh samples was established both 
on the total and vital fraction, before and after density gradient. The data results have been submitted 
and will be available soon (Punjabi 2017).  
In this prospective cohort study we aim to investigate the role of sperm DNA fragmentation analysis in 
selecting the patient who will benefit from intra-uterine insemination (IUI) therapy since IUI is still 
considered the first step in MAR and is performed at a large scale in Belgium and worldwide. 
The overall success rate of IUI remains controversial. On average, reported clinical pregnancy rates are 
only 5–13% per IUI cycle (Bensdorp et al., 2007; Goverde et al., 2000; Guzick et al., 1999; Nyboe 
Andersen et al., 2009; Steures et al., 2004, 2007; The ESHRE Capri Workshop Group, 2009; Tummon 
et al., 1997; Verhulst et al., 2006).  
 
A Cochrane analysis pointed to the position of IUI in male subfertility treatment as being one of the 
most frequently used treatments (Veltman-Verhulst et al., 2016) although its efficacy in the setting of 
mild male/unexplained infertility is yet to be proven. The authors concluded that there was insufficient 
evidence of effectiveness to recommend or advise against IUI.  
In addition, the literature pertaining IUI consists of a broad range of indications for therapy, ranging 
from but not limited to: unexplained fertility, mild male infertility and mild endometriosis. 
Respectively, each of these diagnoses also have slightly different definitions from one study to another, 
thus making a univocal recommendation concerning IUI very challenging. 
Our goal would be to better select patients for IUI, widely considered less invasive and expensive 
therapy than others, as about 50% of the patients entering an IUI program will not become pregnant 
after 4-6 IUI cycles and thus in selected cases (Van Hoof et al., 2009) progression to more advanced 
fertility therapy could be beneficial.  

 
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Study Objectives 
The objective is to test the hypothesis that DNA fragmentation testing can play a diagnostic and thus 
pivotal role in selecting the patient who will benefit from intra-uterine insemination (IUI) therapy 
leading to a higher clinical pregnancy and live birth rate (primary outcomes) compared to the standard 
semen criteria. 
Additionally, DNA fragmentation will be measured both at the time of the diagnostic work-up as at the 
time of insemination with evaluation in the neat and washed specimen, both in the total and vital 
fraction. We aim to assess whether density gradient retrieves high quality motile sperm with little DNA 
damage and where DNA fragmentation testing best fits in the clinical patient pathway (secondary 
outcomes). 
Study Endpoints 
Primary outcomes: 
- 
DNA fragmentation as a predictor of  clinical pregnancy and live birth rate 
Secondary outcomes: 
- 
DNA fragmentation in the total and vital fraction before and after density gradient in the 
diagnostic sample (pre-IUI) 
- 
DNA fragmentation in the total and vital fraction before and after density gradient in the 
therapeutic sample (IUI sample)   
- 
DNA fragmentation in relation to the cumulative clinical pregnancy and live birth rate  
Study Phase & Design 
 
Prospective interventional study 
Duration: 24 months 
Source population: couples with unexplained, mild male infertility, mild endometriosis defined by 
revised American Society for Reproductive Medicine stage I and II  
Single center, university setting, no commercial affiliation 
Study Population 
Inclusion criteria: 
Couples seeking fertility treatment after at least 12 months of unprotected intercourse are eligible. All 
couples underwent basic fertility investigations which included semen analysis, evaluation of menstrual 
cycle, and tubal patency testing (Chlamydia antibody test, hysterosalpingography/HyfoSy or 
laparoscopy).  
All participating couples should provide written informed consent 
Inclusion criteria are  
- 
Unexplained infertility  
o 
We classify couples as having unexplained subfertility when the fertility 
investigations showed at least one patent fallopian tube, a normal ovulatory 

 
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menstrual cycle (26-32), and a normal semen analysis (pre-wash total motile sperm 
count above 10 million). 
- 
Mild male infertility defined as one or more abnormal semenparameters according to the 
WHO 2010 criteria on at least two examinations; diagnosis is determined by most abnormal 
result 
- 
Women: age between 18 and 40 years, BMI between 18,5-30, presence of at least one patent 
tube on hysterosalpingography/Hysterosalpingo foam sonography (Hyfosy) and/or 
laparoscopy, normal uterine cavity (ultrasound, hysterosalpingography or laparoscopy), 
regular menstrual cycle (24-38 days, FIGO recommendations 2011) 
- 
Men: age between 18 and 65 years, BMI between 18,5-30, normal semen analysis or mild 
male subfertility 
- 
Exclusion criteria: double sided tubal disease, severe endometriosis (classified as revised 
American Society for Reproductive Medicine stage III or IV), premature ovarian failure, and 
known endocrine disorders (such as Cushing’s syndrome or adrenal hyperplasia), azoö- or 
necrozoöspermia 
Statistics 
- 
Sample size: 120 patients 
A power analysis was performed to estimate the sample size needed for ROC curve analysis. 
Since there is scarce literature for sperm DNA fragmentation on an IUI sample both before 
and after gradient, we cannot rely on reference values to estimate the area under the curve 
(AUC) or distribution of cases versus controls (kappa).  In function  of a significance of 0,05 
(type I error) and  power of 0,8 (1-type II error), a kappa varying between 1:1 and 1:3 and 
AUC varying between 0,7-0,75 we estimate a minimum of 38 patients and a maximum of 130 
patients should be included. 
 
- 
Diagnostic parameters: sensitivity, specificity, area under receiver operator curve,  predictive 
value, likelihood ratio, odds ratio 
- 
Null hypothesis:  
1) There is no correlation between DNA fragmentation and clinical pregnancy/live 
birth after IUI 
2) Density gradient isn't capable of selecting motile sperm with low DNA 
fragmentation for insemination 
Clinical protocol 
- 
IUI in a spontaneous cycle 
- 
Basal ultrasound and hormonal analysis on cycle day 2 or 3 (estradiol (E2), progesterone (P), 
luteinizing hormone (LH) as well as follicle stimulating hormone (FSH)) 
- 
Cycle monitoring from D10-12 with ultrasound and/or hormonal analysis (E2, P, LH)  
- 
250 microgram hCG or 5000 IU is administered to induce ovulation when a maximum of 2 
dominant follicles are present.  Either Pregnyl, Choragon, Ovitrelle will be used 
- 
IUI is planned between 32-40 hours after HCG administration or ± 24-26 hours after detection of 
spontaneous LH surge 
- 
Both ultrasound and hormonal analysis (E2, P, LH) are done on the day of planning of IUI (day of 
hCG administration or day of LH surge) 
- 
Sperm preparation is performed according to local validated procedures in accordance with the 
2010 WHO criteria. DNA fragmentation analysis by TUNEL assay will be carried out both before 
and after density gradient. 
- 
Patients are advised to rest in the supine position during 15 minutes immediately after IUI 
according to evidence from a RCT (Custers et al. 2009) 
 
- 
Detection of pregnancy 
 
βHCG testing is performed 15-20 days after insemination  
 
A second βHCG testing is performed 7 days later to confirm a positive evolution of pregnancy 
 
In case of a positive evolution of βHCG an ultrasound is performed around 5-6 weeks after 
IUI 
 
Follow-up of pregnancy until birth through staff, telephone or e-mail contact until birth 

 
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- 
Items to be registered 
 
Number of eligible patients (patients who fulfilled inclusion criteria and to whom the study 
was proposed) 
 
Number of drop outs between inclusion and end of treatment 
 
Number of spontaneous pregnancies between inclusion and end of treatment 
 
Number of live births between inclusion and end of treatment 
 
 
Hunault score and modified Hunault-score according to Bensdorp et al. 
 
All Belrap items 
 
Semen analysis methodology checklist according to Björndahl et al. (Appendix) 
 
Patient population characteristics: 
o 
Infertility: duration, primary versus secondary, female/male/mixed/unexplained 
o 
Cause of female infertility: Ovulatory disorder, Anatomical infertility, Endometriosis 
(revised American Society for Reproductive Medicine classifcation) or Adenomyosis 
o 
Possible cause of male infertility: Varicocoele, Cryptorchidism, Male accessory gland 
infections, Germ cell malignancies and testicular microcalcifications, Disorders of 
ejaculation, Idiopathic. Primary spermatic failure, genetic disorders of infertility, 
obstructive azoöspermia, hypogonadism were not reported since azoöspermia is 
considered an exclusion criterium. 
 
- 
Data analysis and Publication policy 
Data analysis and preparation for publication will be done at the University Hospital of Antwerp / 
University of Antwerp.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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