This study aimed to determine the effect of sperm DNA fragmentation (SDF) on the cumulative live birth rate (CLBR) in intracytoplasmic sperm injection (ICSI) cycles in couples with unexplained infertility.
We conducted a prospective study of 145 couples who underwent ICSI cycles for unexplained infertility. Based on the SDF rate, patients were categorized into a low SDF group (SDF ≤30%, n=97) and a high SDF group (SDF >30%, n=48). SDF was assessed using the acridine orange test on density gradient centrifugation prepared samples. The CLBR was calculated as the first live birth event per woman per egg collection over 2 years.
The high SDF group (SDF >30%) showed a significantly lower CLBR (
High SDF was associated with a lower CLBR and a higher miscarriage rate in the ICSI cycles of couples with unexplained infertility.
Infertility affects approximately 15% of couples of reproductive age [
Approximately 25%–80% of couples with unexplained infertility have elevated SDF values [
Previously, intrauterine insemination (IUI) and IVF were the first-line treatments for couples with unexplained infertility [
Two types of assays measure the levels of SDF: one that directly measures the extent of DNA fragmentation using probes and dyes and another that measures the susceptibility of DNA to denaturation, which is higher in fragmented DNA [
A study showed that couples with unexplained infertility had elevated SDF but did not explore clinical correlations with the outcomes of the ICSI cycles [
We conducted a prospective study of 145 couples with unexplained infertility (median age, 30.25±4.33 years) who were undergoing their first ICSI cycles at the tertiary care center attached to our reproductive medicine unit at a medical college. This study was approved by the ethics committee of our institution. Written consent was obtained from all participating couples. A total of 145 ICSI cycles (one ICSI cycle per couple) were divided into two groups based on SDF rates: a low DNA fragmentation group (SDF ≤30%, n=97) and a high DNA fragmentation group (SDF>30%, n=48) [
Couples undergoing their first ICSI cycles for unexplained infertility were included in this study. The diagnosis of unexplained infertility was based on the following criteria: (1) normal ovarian reserve with an antral follicle count ≥8 and anti-Müllerian hormone levels ≥1.5 ng/mL, (2) normal tubal patency and uterine function evaluated by diagnostic laparoscopy and hysteroscopy, and (3) normal semen parameters for the male partner according to World Health Organization (WHO) 2010 criteria [
Patients collected semen samples in sterile, non-toxic containers by masturbation after sexual abstinence of 2–3 days. After 30 minutes of liquefaction, samples were evaluated for count, motility, and morphology according to the WHO 2010 criteria [
The assessment of SDF was done using AOT [
The slides were examined for SDF using a fluorescence microscope (Olympus CX31, Tokyo, Japan) under oil at ×1,000 with an excitation of 450–490 nm. Green fluorescence represented normal intact sperm, whereas red indicated fragmented and denatured sperm. Sperm with orange or yellow heads, as well as those displaying green and red colors simultaneously, were also considered fragmented [
Controlled ovarian stimulation was started from day 3 of the menstrual cycle using recombinant follicle-stimulating hormone (Recagon, MSD; Gonal-F, Merck, Kenilworth, NJ, USA). A gonadotropin-releasing hormone antagonist (Cetrorelix Acetate, Emcure, Pune, India) was administered to suppress the pituitary function when a minimum of one follicle ≥14 mm was seen. Recombinant human chorionic gonadotropin (Ovidrel, Merck) was administered when three or more follicles reached a diameter of ≥17 mm and appropriate serum estradiol values were detected. TVOR was performed 35 hours after triggering with human chorionic gonadotropin.
The recovered oocytes were incubated in culture medium (Onestep; Vitromed) for 1–2 hours at 37°C in an atmosphere of 6% CO2, 5% O2, and the remainder N2. The oocytes were denuded by hyaluronidase (Hyadase 80 IU; Vitromed) at 37°C. The ICSI procedure, as described by Palermo et al. [
According to the Istanbul consensus, day-3 embryos were graded as A, B, and C based on the blastomere number, fragmentation percentage, and multinucleation [
Day-5 blastocysts were graded according to Gardner and Schoolcraft [
The surplus embryos were vitrified on either day 3 or day 5 by the Kitazato vitrification protocol (Kitazato, Japan) [
Similarly, warming of the day-3 or day-5 embryos was done using the Kitazato thawing protocol (Kitazato) [
After oocyte retrieval in patients undergoing ET cycles, daily micronized progesterone was administered vaginally (Crinone 8% gel, Merck, Kenilworth, NJ, USA) and on alternate days intramuscularly (Hald 100 mg, Intas, Ahmedabad, India) until the pregnancy test was confirmed negative, or continued for an additional 3 months if the pregnancy test was positive.
In FET cycle patients, oral estradiol valerate (Evadiol, Intas, Ahmedabad, India) was used in a stepwise increasing dose pattern for preparation of the endometrium. The endometrial lining and thickness were observed regularly prior to the ET. Progesterone was administered in a method like that described in the ET cycles. For a day-3 or day-5 ET, 4 or 6 days of progesterone was administered, respectively.
ET was performed under abdominal guided ultrasound (a maximum of 3 embryos) on either day 3 or day 5, depending on the quality of the embryos and the age of the patient. The embryos were transferred using a soft catheter (Cook, Brisbane, Australia). The serum β-hCG level was obtained 14 days after the transfer to confirm a positive pregnancy. Embryo utilization was calculated as the ratio of the number of embryos transferred and the number of embryos frozen to the total number of embryos formed. The high-quality embryo (grade A) rate at day 3 was calculated as the ratio of grade A embryos at day 3 to the total number of embryos cleaved. An intrauterine sac with the presence of a fetal heartbeat was considered a clinical pregnancy. The implantation rate was calculated as the proportion of gestational sacs determined by ultrasound to the total number of embryos transferred. Miscarriage was defined as a pregnancy loss after detection of an intrauterine pregnancy by ultrasound before 20 weeks. The CLBR was calculated as the first live birth event per woman per egg collection over 2 years.
Data were shown as mean±standard deviation for continuous variables and analyzed using the unpaired Student
When the demographic and embryological characteristics of couples with unexplained infertility were compared between the two SDF groups (low SDF ≤30% and high SDF >30%), similar findings were observed for the ages of the female and male partners, years of infertility, number of previous failed IUI cycles, number of oocytes retrieved, number of metaphase II oocytes, fertilization rates, cleavage rates, embryo utilization rates, number of transferred embryos, and grade A embryo rates at day 3. The only meaningful difference was observed in the number of ET cycles per ICSI. A higher number of ET cycles per ICSI (
Semen parameters such as sperm count, total sperm count, motility, progressive motility, and morphology were similar between the two SDF groups, whereas a significant difference was observed in the SDF rates (
A total of 145 patients underwent 191 ET cycles (both ET and FET). In the low SDF group, 97 patients underwent 97 ICSI cycles and 121 ET cycles, while 48 patients in the high SDF group underwent 48 ICSI cycles and 70 ET cycles. When the clinical outcomes between the two groups were compared, the high SDF group had a significantly lower CLBR (
In the low SDF group, out of 121 ET cycles, 66 (54.54%) were ET cycles and 55 (45.45%) were FET cycles, whereas in the high SDF group, out of 70 ET cycles, 30 (42.85%) were ET cycles and 40 (57.14%) were FET cycles. There was no notable difference in the ET and FET cycles when the two groups were compared (
The day of transfer (day3/day5) and type of transfer (fresh/frozen) were considered as biasing factors. There was no significant difference in the live birth rate of the day-3 or day-5 transfers (
The couples with unexplained infertility were divided into two groups based on live birth outcomes: (1) the positive live birth group and (2) the negative live birth group. These two groups showed significant differences in the ages of both male (
When adjusted for the possible confounders between the positive live birth and negative live birth groups, multivariate logistic regression analysis showed that SDF was a predictor of cumulative live birth in couples with unexplained infertility (
Routine semen analysis plays a salient role in the infertility evaluation of men. However, its role is minor for couples with unexplained infertility since sperm abnormalities at the DNA level cannot be identified by routine methods. SDF, rather than normal semen analysis, has good diagnostic and prognostic capabilities for men with idiopathic infertility based on routine semen parameters [
SDF can occur pre- or post-ejaculation due to various mechanisms, as described by Sakkas and Alvarez [
The main outcome measure of the present study was the CLBR in correlation with SDF in couples with unexplained infertility in ICSI cycles. The high SDF group had a 1.5-fold lower CLBR (
ICSI has been the most favored method for treating couples with well-defined idiopathic infertility [
The negative correlation of SDF with the live birth rate in IVF cycles was established in a recent meta-analysis [
One study reported a significant difference in the live birth rate in IVF cycles with a high SDF and, to a lesser degree, in ICSI cycles; the weaker findings in ICSI cycles can be explained by the fact that there were many fewer patients in the low SDF group (<25%). The same study showed an approximately 12% lower live birth rate in ICSI patients with SDF of 25%–50% compared to those with SDF >50% [
SDF was positively correlated with the miscarriage rate in this study at a threshold of 30%. Spontaneous abortion rates were higher in ICSI cycles with SDF>30%, as reported by Zini et al. [
To some extent, the effect of SDF on the clinical outcome depends on the quality of the oocyte [
The SDF, fertilization, and cleavage rates showed no notable differences between the live birth groups. In the positive live birth group, the grade A embryo rate was higher (
AOT is an established method for assessing the integrity of sperm DNA in infertile men [
The AOT method is simple, inexpensive, and convenient to use routinely in-house. The principle of AOT is similar to that of the sperm chromatin structural assay (SCSA) except for the number of sperm counted. In this study, a trained and technically skilled in-house embryologist evaluated the slides for SDF. We have been using the AOT method to assess SDF since 2012 for various research projects [
The percentage of couples with high SDF in this study was approximately 33% of all couples with unexplained infertility. The low percentage compared to other studies may have been due to the use of prepared sperm samples to evaluate SDF rather than raw semen samples [
Despite the valuable results obtained in the study, the authors recognize its limitations. The sample size was small because only couples with unexplained infertility who underwent ICSI cycles were included. The AOT method may not be as robust as the gold-standard SCSA method but, as already mentioned, the AOT method is simple, inexpensive, and comparable to the SCSA method. We were unable to calculate the blastulation rate as some patients underwent both day-3 and day-5 ET cycles. Finally, SDF is a contributing factor along with other confounders, not an independent predictor of CLBR in the ICSI cycles of couples with unexplained infertility.
In conclusion, SDF negatively influenced the CLBR, and a high SDF was associated with a higher miscarriage rate in the ICSI cycles of couples with unexplained infertility. These findings suggest that there is a need to evaluate SDF prior to ART cycles in couples with unexplained infertility to enable better counseling.
We would like to acknowledge all staff members of the reproductive medicine and surgery department and the couples who participated in the study.
No potential conflict of interest relevant to this article was reported.
Conceptualization: DR. Data curation: DR. Formal analysis: DR. Methodology: DR. Project administration: DR, SB. Visualization: DR, KVRS. Writing–original draft: DR. Writing–review & editing: all authors.
(A) Percentage of fresh embryo transfer (ET) and frozen embryo transfer (FET) cycles and (B) day 3 and day 5 embryo transfer cycles in the low sperm DNA fragmentation (SDF; ≤30%) and high SDF (>30%) groups.
Demographic and embryological characteristics of the couples with unexplained infertility couples who underwent ICSI cycles
Characteristics | SDF ≤30% | SDF >30% | |
---|---|---|---|
No. of patients | 97 | 48 | |
Female age (yr) | 30.15±4.27 | 30.44±4.48 | 0.705 |
Male age (yr) | 34.40±4.64 | 34.85±4.03 | 0.567 |
Year of infertility | 2.98±1.52 | 3.06±1.47 | 0.785 |
No. of previous failed IUI cycles | 1.94±0.65 | 2.06±0.69 | 0.334 |
No. of oocytes retrieved | 14.83±5.60 | 13.66±4.41 | 0.209 |
No. of MII oocytes | 12.60±5.38 | 11.35±4.26 | 0.162 |
Fertilization rate | 84.08±14.62 | 87.47±14.30 | 0.188 |
Cleavage rate | 82.19±15.69 | 84.77±16.74 | 0.364 |
Embryo utilization rate | 65.82±22.33 | 70.84±22.42 | 0.206 |
Good quality embryo rate at day 3 | 41.18±20.87 | 43.15±23.66 | 0.610 |
No. of embryos transferred | 2.11±0.59 | 2.09±0.56 | 0.818 |
No. of embryo transfer cycles | 1.25±0.48 | 1.46±0.54 | 0.018 |
Values are presented as mean±standard deviation.
ICSI, intracytoplasmic sperm injection; SDF, sperm DNA fragmentation; IUI, intrauterine insemination; MII, metaphase II.
Comparative analysis of semen parameters according to SDF group
Parameter | SDF ≤30% | SDF >30% | |
---|---|---|---|
Sperm count (×106/mL) | 39.06±11.92 | 35.66±12.78 | 0.117 |
Total sperm count (×106) | 94.85±37.49 | 86.91±36.39 | 0.228 |
Motility (%) | 58.79±8.52 | 58.47±8.24 | 0.833 |
Progressive motility (%) | 45.64±7.10 | 46.37±7.23 | 0.228 |
Morphology (%) | 5.18±0.93 | 5.20±0.89 | 0.889 |
SDF rate | 14.19±8.02 | 53.81±16.28 | <0.001 |
Values are presented as mean±standard deviation.
SDF, sperm DNA fragmentation.
Clinical outcomes of patients with unexplained infertility who underwent ICSI cycles
Clinical outcome | SDF ≤30% | SDF >30% | |
---|---|---|---|
Total embryo transfer cycles (n=191) | n=121 | n=70 | |
Implantation rate | 38.61 (95/246) | 29.10 (39/134) | 0.063 |
Cumulative pregnancy rate | 75.25 (73/97) | 66.66 (32/48) | 0.276 |
Cumulative live birth rate | 60.82 (59/97) | 41.66 (20/48) | 0.029 |
Miscarriage rate | 19.17 (14/73) | 37.5 (12/32) | 0.045 |
Fresh embryo transfers (n=96) | n=66 | n=30 | |
Implantation rate | 34.72 (50/144) | 20.00 (13/65) | 0.031 |
Clinical pregnancy rate | 66.66 (44/66) | 36.66 (11/30) | 0.005 |
Live birth rate | 54.54 (36/66) | 23.33 (7/30) | 0.004 |
Miscarriage rate | 18.18 (8/44) | 36.36 (4/11) | 0.191 |
Frozen embryo transfers (n=95) | n=55 | n=40 | |
Implantation rate | 44.11 (45/102) | 37.68 (26/69) | 0.402 |
Clinical pregnancy rate | 52.72 (29/55) | 52.50 (21/40) | 0.982 |
Live birth rate | 41.81 (23/55) | 32.50 (13/40) | 0.355 |
Miscarriage rate | 20.68 (6/29) | 38.09 (8/21) | 0.176 |
Values are presented as percent (positive number/total number).
ICSI, intracytoplasmic sperm injection; SDF, sperm DNA fragmentation.
Stratification of the biasing factors between positive live birth and negative live birth groups
Biasing factor | Positive live birth group | Negative live birth group | |
---|---|---|---|
Day of transfer (day 3/day 5) | 79 (30/49) | 66 (33/33) | 0.145 |
Type of transfer (fresh/frozen) | 79 (38/41) | 66 (28/38) | 0.494 |
Demographic and embryological characteristics of couples with unexplained infertility subdivided into the live birth groups
Characteristics | Positive live birth group | Negative live birth group | |
---|---|---|---|
No. of patients | 79 | 66 | - |
Female partner’s age (yr) | 29.56±3.77 | 31.08±4.81 | 0.034 |
Male partner’s age (yr) | 33.77±3.92 | 35.48±4.85 | 0.020 |
SDF rate | 24.28±19.07 | 30.92±24.53 | 0.068 |
No. of MII oocytes | 11.90±5.14 | 11.73±5.36 | 0.846 |
Fertilization rate | 86.78±12.67 | 83.31±16.43 | 0.153 |
Cleavage rate | 84.99±14.16 | 80.71±17.86 | 0.110 |
Embryo utilization rate | 72.43±22.66 | 63.96±21.75 | 0.023 |
High-quality embryo rate | 45.13±21.16 | 37.88±21.99 | 0.045 |
No. of embryos transferred | 2.09±0.59 | 2.24±0.56 | 0.088 |
No. of embryo transfer cycles | 1.28±0.50 | 1.36±0.52 | 0.347 |
Values are presented as mean±standard deviation.
SDF, sperm DNA fragmentation; MII, metaphase II.
SDF as a predictor of cumulative live birth and miscarriage in ICSI cycles of unexplained infertility couples
Clinical outcome | Adjusted OR | 95% CI | Confounder adjusted | |
---|---|---|---|---|
Cumulative live birth | 0.984 | 0.968–1.000 | 0.047 | Female partner’s age, embryo utilization rate, high-quality embryo rate |
Miscarriage | 1.005 | 0.985–1.025 | 0.621 | Female partner’s age, embryo utilization rate, high-quality embryo rate |
SDF, sperm DNA fragmentation; ICSI, intracytoplasmic sperm injection; OR, odds ratio; CI, confidence interval.