An analysis of 2,566 cycles Premature progesterone rise negatively correlated with live birth rate in IVF cycles with GnRH agonist: An analysis of 2,566 cycles Fertility and Sterility Vol. 98, No. 3, September th October 2012 Tuesday Infertility Journal reading R4 孫怡虹 /Dr. 蔡永杰

Serum P level in IVF cycles ↑ On the day of hCG administration: Not uncommon – Associated with: poor oocyte quality, low fertilization rate, adverse pregnancy outcome – Induced by inappropriate LH elevation (premature luteinization) After the introduction of GnRH analogue – Inhibit LH elevation  P level rise would be rare – Subtle rise is still about 5%–71%: Premature progesterone rise (PPR)

Premature progesterone rise The influence on pregnancy outcomes: controversial ↑ Published papers since the 1990s: – Negative effect vs. Positive correlation – Systematic review and meta-analysis (2007, Venetis et al.): No correlation – Retrospective study (N > 4000, 2010, Bosch et al.): P level >1.5 ng/Ml In both GnRH agonist & antagonist cycles Associated with ↓ ongoing pregnancy rate

Premature progesterone rise Underlying mechanism to influences clinical outcomes: elusive – Premature of endometrium  a detrimental factor to the endometrial receptivity – Compromised quality of oocytes

Premature progesterone rise Pathogenesis and etiology: poorly understood 1)During controlled ovarian stimulation (COS)  multiple follicles (each one producing a normal amount of P)  Accumulated P production 2)Overdose of exogenous gonadotropin to promote: cholesterol  P (Acting on granulosa cells) 3)Premature luteinization, as the consequence of subtle LH rise (Even with GnRHa) 4)Poor ovarian response with increased LH sensitivity

Hypothesis If PPR is harmful to clinical outcomes, either triggering hCG in advance or freezing all embryos should be recommended to avoid the adverse consequences

Aim Retrospective analysis To quantify the incidence of PPR – In a large cohort of patients (n = 2,566) – Undergoing COS with GnRH agonist long or short protocol ?To investigate the relationship: Serum P level  Probability of live birth ?Possible mechanism: PPR affects live birth rate ?Possible factor(s) related to the occurrence of PPR

MATERIALS AND METHODS

Non-interventional retrospective analysis Reproductive Medicine Center of the Women and Children Hospital of Guangdong Province January 1, 2002 ~ December 30, 2007 – Women who started their first IVF/ICSI cycles with GnRH agonist treatment: All IVF/intra-cytoplasmic sperm injection (ICSI) cycles – All with Controlled Ovarian Stimulation – In the case of P level rise  No fresh ET canceled F/u through December 2011 – For clinical data of frozen ET

3,034 patients  Excluded: 468 cycles (No embryo formed or all embryos were frozen owing to OHSS or other personal reasons) Long protocol (n = 1,719)  Short protocol (n = 847) Short protocol – 2002 ~ 2004, Short protocol: prevalent (650/733, 88.7%)  Long protocol: PCOS patients or some potential high responders Long protocol – 2005 ~ 2007, Long protocol: dominant (1,636/1,833, 89.3%)  Short protocol: potential poor responders or patients refusing long protocol for personal reasons

Controlled Ovarian Stimulation Protocol Long protocol: – Triptorelin depot mg or 1.3 mg  Down regulation – Recombinant FSH  Ovarian stimulation Short protocol: – Triptorelin 0.1 mg  In cycle day 2 – rFSH  From day 3

Controlled Ovarian Stimulation Protocol Follicle growth monitoring: – Serum E2, P, and LH level measurements – Vaginal ultrasound investigation  ≥ 2 follicles: size of ≥ 18 mm  35 hours later  10,000 IU hCG for the oocyte retrieval

Embryo Culture, Transfer, Freezing and Thawing Cultured in Quinnes sequential culture media – Transferred on day 3: ≤ 3 Embryos  Luteal support: 20–40 mg P in oil – Surplus embryos frozen: ≥ 4 cells, < 25% fragmentation For frozen ET: – Transferred: ≤ 3 frozen-thawed survival embryos – In a natural or hormonal replacement cycle

Hormone Assays Serum: 8–9 a.m. at each time point FSH, LH, E2, P  analyzed by radioimmunoassay Intra- vs. inter-assay coefficients of variation: – LH = 5.8% vs. 9.1% – FSH = 5.6% vs. 7.8% – E2 = 8.2% vs. 9.3% – P = 7.0% vs. 9.6% Progesterone: – Sensitivity = 0.1 ng/ml – Range of measurement = 0.1–40 ng/mL

Outcome Variables Primary outcome variable: Live birth rate Secondary outcome variables: – Clinical pregnancy rate: Presence of FHB on ultrasound at 7 weeks' pregnancy – Implantation rate: Number (Gestational sacs seen on the ultrasound) Total number (Embryos transferred)

Statistical Analysis Patients  divided into 6 groups: – According to Serum [P] (ng/mL) on the day of hCG administration: – ≤ 0.9, 1.0–1.2, 1.3–1.5, 1.6–1.8, 1.9–2.0, >2.00 Bosch method (Trend analysis)  To determine the cutoff value of PPR Odds ratio (OR) of live birth rate (each P interval  preceding interval)

Logistic regression analysis: – Parameters possibly related to live birth rate ( x 14) – In which P level, age, and the number of high- quality embryos transferred were categorized Multivariate analysis: for assessing Factors related to PPR

RESULTS

General Information: Baseline & cycle characteristics of the patients with long or short protocol

Relationship between serum P levels & live birth rates

Live birth rates according to serum P levels “Nonlinear Relationship”

Live birth rates according to serum P levels Suggest the Cutoff value to define PPR (Negative impact of P on live birth rate) “Nonlinear Relationship”

Incidence of PPR Long protocol: 22.86% (393/1719) Short protocol: 27.63% (234/847)

Clinical outcomes (With  Without PPR) Clinical pregnancy rate – Long protocol: OR 0.74, P=0.004 – Short protocol: OR 0.84, P=0.002 Implantation rate – Long protocol: OR 0.73, P=0.000 – Short protocol: OR 0.71, P=0.005 Live birth rate – Long protocol: 40.65/29.77(%), OR 0.73; P=.004 – Short protocol: 30.18/23.50(%), OR 0.78; P=.083

Logistic regression analysis For potential factors (might influence live birth rate) Confirmed: P level did have a negative effect on live birth rate in both (long/short) protocols

Effect of PPR on Live Birth Rate in Frozen ET Cycles 956 frozen ET cycles (670 patients) in long protocol – P ≤ 1.2 ng/mL  live birth rate: 197/672 (29.31%) – P > 1.2 ng/mL  live birth rate: 72/284 (25.35%) – No statistical significance observed (P=0.213) 483 frozen ET cycles (324 patients) in short protocol – P ≤ 2 ng/mL  live birth rate: 80/322 (24.84%) – P > 2 ng/mL  live birth rate: 39/161 (24.22% – No statistical significance observed (P=0.881)

Multivariate regression analysis of factors related to premature P rise

DISCUSSION

Relationship between PPR and clinical outcomes Controversy – Distinct definition of PPR (0.9 ~ 2.0 ng/mL) – Different statistic methods used – Different characteristics of patients recruited – Retrospective nature of studies analyzing the relationship between PPR and clinical outcomes

Relationship between PPR and clinical outcomes Not linear in this study (consistent with Bosch et al.'s report)  Trend analysis  Negative relationship: PPR  live birth rate  Cutoff value: 1.2 ng/mL in long protocol & 2.0 ng/mL in short protocol (  1.5 ng/mL)  Analyse Live birth rate (  Ongoing pregnancy rate)  Patients were much younger (31.1 vs years)  Different methods of progesterone measurement

Relationship of PPR and clinical outcomes in GnRH agonist long protocol Some studies failed to observe negative correlation: – Limited number of patients recruited – Relatively low threshold used to define PPR Present study: Largest number of patients (n = 1,719) with long protocol  strengthening the conclusion

In this study: 1)P level: Short protocol > Long protocol – Flare-up effect of GnRH agonist  relatively ↑ LH – LH might also be involved in the occurrence of PPR 2)Clinical pregnancy rate (P=.002) & Live birth rate (P=.083) ↓ when P level >2 ng/mL 3)Normal & poor responders (former predominant) 4)↑ Number of patients involved 5)Cutoff value to define PPR: 2.0 ng/Ml Relationship of PPR and clinical outcomes in short protocol

The distinct cutoff values determined in the present study 1)The characteristics of patients in long or short protocol were different from each other, more poor responders being included in short protocol 2)The cycle properties of the two protocols are different, with the LH level in the follicle phase being much higher in short protocol 3)The number of patients included in short protocol was relatively few, which might have introduced a bias

Logistic regression analysis in the present study To exclude interference from confounding factors – Difficult in retrospective study & Prospective studies 13 confounding factors  Confirmed: PPR was a negative predictor of live birth rate  Low pre ovulatory serum P  A dominant predictor of clinical pregnancy

How PPR affects live birth rate ? Compromised oocyte quality ?  If it’s the main reason  Triggering hCG in advance would be the first choice (avoid the adverse effects of PPR on the quality of oocytes) Decreased endometrial receptivity ?  If it plays a key role  freezing embryos would be a better choice (triggering hCG in advance: would compromise the overall quality of the oocyte cohort when most of the trailing follicles are still small)

How PPR affects live birth rate ? Frozen ET cycles: long protocol (n = 956) and short protocol (n = 483)  Live birth rates: Not significantly different between the groups with or without PPR  PPR may negatively affect live birth rate through decreased endometrial receptivity Based on a donor oocyte IVF model, Melo et al.  Pregnancy rates of recipients: Not influenced by the P levels of donors  ↑ P did not deteriorate the quality of oocytes

Gene expression profile of endometrium P level >1.5 ng/mL vs. normal  obviously different van Vaerenberg et al. Difference maintained until implantation occurred Labarta et al.  ↓ Endometrial receptivity  may be a cause of ↓ live birth rate  For Case of P rise on the day of hCG administration: (The most appropriate choice to avoid the negative effect): cancel fresh ET & freeze all embryos (rather than triggering hCG in advance)

Underlying mechanism of the occurrence of PPR Compare multiple variables: Groups with  without PPR – Factors showing difference  the reason of PPR – Simple/weak to distinguish causal/non-causal correlation – Can’t reflect the interaction among variables Logistic regression in this stidy  Analyze which factors were involved in PPR  P level (continuous variable) be categorized into 2 groups  minify the influence of some factors

Possible explanations of the occurrence of PPR 1.Multiple follicle growth 2.Overdose of exogenous gonadotropin 3.Premature lutineization  Partly explain the reasons from limited facets  Multivariate regression analysis in this study: all contributed to PPR (  Hugues et al.'s conclusion) 4.Poor ovarian response  Not that convincing (women with lower basal FSH level  inclined to have PPR)

Multivariate regression analysis Included Baseline characteristics – (Basal FSH, E2 level, BMI, Age) – Dose of gonadotropin administered – Patients' ovarian response (Number of oocytes retrieved, E2 level on the day of hCG administration)  Make the conclusion more reliable & comprehensive Influence

Summary In these 2,566 patients undergoing their first IVF/ICSI cycles treated with GnRH agonist long/short protocol… PPR  Negatively correlated with live birth rate in fresh ET cycles  No adverse impact on frozen ET observed  PPR may have deleterious effects on endometrial receptivity

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