This was a retrospective cohort study of women undergoing their first in-vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) and fresh embryo transfer (ET) cycle at Reproductive Medicine Center of Xiangya Hospital from 2015 to 2017. Data were collected through the electronic records. This study was approved by the Ethics committee of Xiangya Hospital. The study population was divided into three groups based on the results of MTHFR C677T genotyping (MTHFR 677CC, MTHFR 677CT, and MTHFR 677TT).
Women undergoing their first IVF/ICIS and fresh ET cycle; The couple are Chinese Han nationality; 1~2 good embryos transferred to the patients at Day 3; 18 kg/m2 ≤ BM I≤ 25 kg/m2; Maternal age ≤ 45 years.
Abnormal karyotype in any of the couple. Thyroid dysfunction. Severe Hydrosalpinx. Intrauterine adhesions. Myoma>5cm. Donor cycles.
Controlled ovarian stimulation (COS) was performed using recombinant FSH (rFSH) with or without human Menopausal Gonadotrophin (HMG), or urinary FSH (uFSH) with or without HMG, under pituitary suppression by Gonadotrophin releasing hormone analogue (GnRHa). The choice of starting gonadotrophin (Gn) was based on maternal age, BMI, Basal FSH concentration, AMH concentration, then Gn dose can be adjusted according to patients’ response during COS. The COS protocol was decided according to the physicians’ clinical experience and ART indications. For GnRHa long protocol, Triptorelin (Diphereline, France; 0.25mg/d subcutaneous injection) starting from the middle luteal phase of the previous cycle. After two weeks, pituitary suppression was verified (follicular diameter < 5 mm, endometrial thickness < 5 mm, serum estradiol < 50 pg/mL, serum FSH < 5 IU/L, serum LH < 5 IU/L) before starting COS. For GnRHa short protocol, Triptorelin was started at a subcutaneous dose of 0.25mg/d at Day 2, COS was started at Day 3. For ultra-long protocol, the first injection of Leuprorelin (Livzon Corp., Shanghai, China, 3.75mg, subcutaneously) was given to patients, 4 weeks later, the second Leuprorelin (, 3.75mg, subcutaneously) was given, 3-4 weeks later, COS was started if down-regulation was satisfied. Follicular development was monitored by transvaginal ultrasound and serum estradiol (E2). Measurements performed every 2-3 days from stimulation day 5-6. Ovulation was triggered by injecting 10,000 international units (IU) of hCG (Livzon Corp., Shanghai, China) intramuscularly when at least 1 follicle reached 18 mm, or at least 3 follicles reached 16 mm. After 36 hours of hCG injection, ultrasound guided transvaginal oocyte aspiration (OPU) was performed. Luteal phase support was performed at the day of OPU. Embryos were transferred at Day 3. All the patients were supplemented with 800 mg folic acid from embryo transfer (ET) day to 12 gestational weeks if pregnancy is established.
Sample collection and MTHFR C677T genotyping
Peripheral blood was collected from every women before ART treatment. Genomic DNA was extracted from blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the genotype of MTHFR C677T.
The categorical variables were presented as frequencies and percentages, the continuous variables were presented as means and standard deviations (SDs) or interquartile ranges (IQRs) depending on whether the data distribution was normal. . We compared categorical variables between groups using χ2 testing, and we compared continuous variables using analysis of variance (ANOVA) or the Kruskal-Wallis test according to distribution type. We evaluated the relationship of MTHFR 677 genotype and live birth using logistic regression model with and without adjusting the confounding factors that may have expected influence on our outcome. We used 3 models in total, including model 1, unadjusted; model 2, adjusted for age and BMI, for model 3, adjusted model 2 for infertility type, stimulation protocol, ART indication, miscarriage history, and endometrial thickness.
All statistical analyses were 2-sided, and we considered a p-value of less than 0.05 to be statistically significant. The statistical analyses were performed by SPSS (version 25, Chicago, USA). Hardy-Weinberg equilibrium (HWE) was examined using the StataSE software (version 15.0, Stata Corp., USA). Deviation from HWE was confirmed if p-value < 0.05.
Outcomes and definitions
The primary outcomes were live birth rate, and cumulative live birth rate per stimulation cycle. The secondary outcomes including laboratory outcomes ( number of oocytes, number of 2PNs, number of available embryos, number of good embryos) and clinical outcomes (chemical pregnancy rate, clinical pregnancy rate, implantation rate, preclinical pregnancy loss rate, miscarriage rate).
Chemical pregnancy cases were defined as increased serum HCG levels (>10 IU/L) at 12 days after embryo transfer. clinical pregnancy was confirmed by ultrasonographic visualization of gestational sacs 4–5 weeks after embryo transfer. Implantation rate was calculated as the gestational sac number divided by the transferred embryos number. We defined preclinical pregnancy loss as cases who was chemical pregnancy but not reached clinical pregnancy. Miscarriage was defined as spontaneous clinical pregnancy loss before 24 gestational weeks. Live birth was defined as birth of at least one newborn after 24 weeks’ gestation that exhibits any sign of life. We also reported the cumulative live birth a stimulation cycle at 24 months after their first oocyte retrieve cycle.