Ovarian Reserve

Clinical information from the subject’s electronic medical record was abstracted by the research nurses. All subjects underwent an evaluation for infertility which included a follicle-stimulating hormone (FSH) level drawn on the third day of the menstrual cycle to assess ovarian reserve. After completion of the standard infertility work-up, each subject was given an infertility diagnosis by their reproductive endocrinologist according to the Society for Assisted Reproductive Technology (SART) definitions. SART diagnoses consisted of male factor infertility which included poor semen quantity/quality; female factor infertility which included endometriosis, diminished ovarian reserve, tubal and uterine disorders; other causes and unexplained infertility.
Upon completion of the infertility evaluation, subjects underwent one of three IVF treatment protocols used at the MGH Fertility Center. The three IVF treatment protocols were: (1) Luteal phase GnRH-agonist protocol using low, regular and high-dose leuprolide (Lupron), in which pituitary desensitization was begun in the luteal phase; (2) Follicular phase GnRH-agonist/Flare protocol, in which Lupron was begun in the follicular phase on day 2 of menses at 20 units and decreased to the standard dose of five units on day 5; and (3) GnRH-antagonist protocol, in which GnRH-antagonist was begun when the lead follicle reached 14 mm in size. All cycles were preceded by a cycle of oral contraceptive pills unless contraindicated. On day 3 of induced menses, exogenous gonadotropins [FSH (Gonal-F, Follistim, Bravelle)] and/or Human Menopausal Gonadotropin [hMG (Repronex, Menopur)] were initiated. In the luteal phase GnRH-agonist protocol, Lupron dose was reduced at, or shortly after, the start of ovarian stimulation with FSH/hMG. FSH/hMG and GnRH-agonist or GnRH-antagonist was continued to the day of trigger with Human Chorionic Gonadotropin (hCG), 36 h before oocyte retrieval.

The primary outcome measures for the study were the cumulative probability of conception by 6 menstrual cycles, by 12 menstrual cycles, and relative fecundability. There were no secondary outcomes, but planned exploratory analyses examined associations between levels of AMH and the primary outcomes among age subgroups and between parity subgroups.
The biomarkers of ovarian reserve were considered as categorical variables where informed choices for cut-points were available. It was hypothesized that the relationship between AMH and fertility would be non-linear. After exploring clinical AMH cut off values of 0.4ng/ml, 0.7ng/ml, and 1.0ng/ml, the middle cut-off value of 0.7ng/ml was selected based on previous research.^{13 (link)} The 90^th percentile was selected as the upper level AMH cut-off value (8.5 ng/ml). The clinical value of 10mIU/ml was selected a priori as the serum FSH cut-off value.^{14 (link)} For urine, the corresponding FSH value is 11.5 mIU/mg creatinine, as documented previously.^{13 (link)} Inhibin B was modeled as a continuous variable, as no clinical cut-off values were available.
Non-parametric bivariate analyses were used to compare median biomarker levels by participant characteristics. Because women did not all enter at the same point during their attempts to conceive and some women withdrew, started fertility medications, or were lost to follow up, the cohort was analyzed using a discrete-time Cox proportional hazards model. Time was menstrual cycles at risk for pregnancy (pregnancy attempt cycle). Pregnancy attempt cycle was determined from the time a woman started trying to conceive, not from the time of enrollment. Attempt cycle at enrollment was defined by the pregnancy attempt cycle (usually cycle 1, 2, or 3) in which the woman began participation (completed diaries or baseline questionnaire). Women were censored at the time they withdrew, started fertility medications, or were lost to follow up. Thus, cycles from enrollment to censoring were included in the analysis. As time in these models is measured by menstrual cycles (and not chronologic time) the hazard ratios (HRs) are commonly referred to as fecundability ratios, which are the relative probability of pregnancy in a given cycle for the exposed relative to the reference group. In such models an HR less than one suggests reduced fecundability in the exposed (or non-referent) group.
The Cox proportional hazard models were then used to calculate the cumulative probability of conceiving (with 95% confidence intervals) at 6 and 12 cycles of attempt for each biomarker level. All models adjusted for age (3 categories: <35, 35–37, 38–44)¹⁵ , body mass index (4 categories: <18.5, 18.5–24.9, 25–29.9, ≥30 kg/m²)¹⁶ , race (White: yes/no), current smoking status (yes/no), and hormonal contraceptive use in the preceding year (yes/no). Adjusted Kaplan Meier curves with 95% confidence intervals were also constructed. The predicted probabilities and Kaplan Meier curves were calculated by setting all of the covariates to the mean of the cohort. Planned subgroup analyses were conducted by age and parity. To test for interaction by age and parity, a likelihood ratio test was used to compare the fit for the model without the interaction term to the model including the interaction term. In addition, post hoc sensitivity analyses were conducted by creating additional Cox models to assess different cut-off values and to evaluate potential biases.
A sample size of 750 women was selected based on an a priori power analysis. A 10% loss to follow-up, 70% pregnancy rate in the control group, a 57% pregnancy rate by 6 months in the diminished ovarian reserve group, and 80% power at a type I error rate of 0.05 was conservatively presumed based on the pilot study. ^{13 (link)} SAS (version 9.3) and R (version 3.3.0) were used for statistical analysis. All testing was two-sided. P-values less than 0.05 were considered statistically significant; there was no adjustment for multiple comparisons.