We conducted a retrospective cohort study. The study was carried out in the Department of Reproductive Medicine of a university-level teaching hospital in South India. All couples who underwent ART for non-male factor infertility and resulted in a fresh or frozen embryo transfer between July 2010 and June 2018 were included in the study. The type of ART treatment included either intracytoplasmic sperm injection (ICSI) or a combination of in vitro fertilization (IVF) and ICSI. As this was a retrospective study, ethics committee approval was not required.
We included all couples who underwent ART, and the male partner had a normal diagnostic semen analysis. Semen volume was determined using wide-bore volumetric pipette, and motility was assessed by wet slide preparation. Concentration was assessed using Neubauer’s counting chamber, and morphology was assessed using prestained slides and graded by Kruger’s criteria. The analysis of semen parameters was based on lower reference ranges as defined by the WHO 2010 criteria . We excluded the following couples: (i) female partner’s age more than 40 years, (ii) poor response (less than or equal to 4 oocytes obtained on oocyte retrieval), and (iii) cycles where cryopreserved semen sample was used. We included only a single ART cycle for each couple for analysis of outcomes.
We divided the cases into two groups based on the age of the male partner. Group I included couples where the male partner’s age was less than 40 years and was chosen to be the reference group. Group II included couples with male partner’s age more than or equal to 40 years.
We used conventional long GnRH agonist, GnRH antagonist, or ultralong protocols depending on indication for ART. Controlled ovarian hyperstimulation was done using recombinant FSH (between 100 and 300 IU), and follicular monitoring was carried out by serial transvaginal ultrasound. An hCG trigger of 5000 IU was administered when at least 3 follicles ≥ 17 mm were seen on ultrasound. Oocyte retrieval was done after 35 h, after the trigger. Between one and three embryos were transferred either at cleavage (day 2 or 3) or blastocyst stage (day 5). Luteal support was given with intravaginal micronized progesterone, 400 mg twice daily, and intramuscular progesterone (100 mg) was administered twice weekly. Pregnancy was confirmed by checking serum beta hCG levels on day 18 after oocyte retrieval.
In cycles where an elective cryopreservation of embryos was done, a frozen embryo transfer was planned after artificial preparation of the endometrium was done using escalating doses of oral estrogen valerate and intravaginal progesterone. The transfer was planned according to the day of embryo cryopreservation. Luteal support was the same as that given for fresh cycles. The serum beta hCG was checked on the 18th day of starting progesterone.
Information regarding clinical and laboratory variables such as age, indication, oocyte numbers, embryo quality, number of embryos transferred, type of transfer (fresh vs. frozen), and day of transfer (cleavage vs. blastocyst) were obtained from the departmental ART database. The pregnancy outcomes were collected from the women through e-mails and telephone contacts. Collected data were entered in SPSS, and data were analyzed using STATA, version 21.0 (Statacorp).
The primary outcome was live birth per embryo transfer. Live birth was defined as delivery after 24 completed weeks of gestation. Secondary outcomes included clinical pregnancy rate, miscarriage rate, fertilization rate, embryo development rate, and blastulation rate.
Clinical pregnancy was defined as presence of a gestational sac on ultrasound between 6 and 8 weeks of gestation. Miscarriage rate was defined as the number of spontaneous losses of the fetus or the absence of cardiac activity prior to 24 weeks divided by the number of clinical pregnancies. Fertilization rate was defined as the total number of fertilised oocytes divided by the total number of inseminated or injected oocytes. Embryo development rate was defined as the total number of embryos which have undergone cleavage divided by the total number of fertilised oocytes. Blastulation rate was defined as total number of blastocysts divided by the number of cleaved embryos.
Data were reported as mean (SD) for continuous variables and frequency (percentages) for categorical variables. t test was used to compare both groups. To find out the association between two categorical data, the chi-square test was used. Fertilization rate, embryo development rate, and blastulation rate were compared between the age groups using proportion test. Dichotomous outcomes (live birth, miscarriage, and clinical pregnancy) were analyzed by simple logistic regression. Multiple logistic regression models were constructed to control for potential confounders such as female age, female body mass index, indication for ART, day of transfer (cleavage vs. blastocyst), and type of transfer (fresh vs. frozen) and assess the association between paternal age and outcomes. The effect is reported as odds ratio (OR) with 95% confidence interval (CI). Data were analyzed using SPSS, version 21.0 (Armonk, NY: IBM Corp).