Natural Cycle vs. Modified Natural Cycle vs. Artificial Cycle Protocol for Endometrial Preparation.

The Effectiveness and Safety of the Three Endometrial Preparation Protocols for Frozen Embryo Transfer Natural Cycle, Modified Natural Cycle and Artificial Cycle: a Randomized Controlled Trial

Fresh embryo transfer is a routine procedure in vitro fertilization (IVF) treatment. Since the first live birth after an IVF-FET (frozen embryo transfer), recent years have seen a dramatic rise in the number of FET cycles. Three endometrial preparation protocols for frozen-thawed embryo transfer, including artificial, natural, modified natural protocol, have been studied and applied to maximize treatment outcomes. However, those methods are being applied empirically as their efficacy and safety are yet to be determined. The objective of this study is to compare the effectiveness and safety of those protocols.

This trial will be conducted at My Duc Hospital, Ho Chi Minh City, Viet Nam. Women who are potentially eligible will be provided information about the trial as long as their stimulation cycles are initiated. Screening for eligibility will be performed by treating physicians on day 2 of the menstrual cycle in the subsequent frozen embryo transfer cycles. Patients will be provided a copy of the informed consent documents. Written informed consent will be obtained by the investigator from all women before the enrolment. Women will be randomized (1:1:1) to AC (artificial cycle) or NC (natural cycle), or mNC (modified natural cycle) protocols using block randomization with a variable block size of 6 or 9 by an independent study coordinator via telephone, using a computer-generated random list (block size of 6, or 9).

Artificial protocol The endometrium is prepared using oral estradiol valerate (Valiera®; Laboratories Recalcine) 8 mg/day, ranging from the second or fourth menstruation day. The endometrial thickness will be monitored from day six onwards, and vaginal progesterone (Utrogestan®; Besins) 800 mg/day will be initiated when endometrial thickness reaches ≥7 mm. Estradiol exposure must be lasting for ≥9 days before progesterone administration. Embryo transfer will be scheduled by the time of the initiation of progesterone and embryo stages. In cases where a dominant follicle emerged, serum LH and progesterone will be determined to rule out luteinization. If LH concentrations are <13 IU and progesterone levels <15 nmol/l, luteinization will deem not to have occurred, and FET was performed.

Natural protocol The first ultrasound scan will be performed on the second to the fourth day of the menstrual cycle to identify any problem related to the patient's uterus or adnexa. The second ultrasound will be performed on the sixth day of the cycle. Daily ultrasound and serum estradiol and LH level evaluation will be performed when the mean diameter of the dominant follicle of ≥14 mm. LH surge initiation is defined as a concentration of 180% above the latest serum value available in that patient with a continued rise thereafter to a level of 20 IU/l or more detected by the ECLIA method (Roche Cobas® E 801, Roche Diagnostics, Germany). Embryo transfer will be scheduled by the time of the initiation of LH and embryo stages.

modified Natural protocol The first ultrasound scan will be performed on the second to the fourth day of the menstrual cycle to identify any problem related to the patient's uterus or adnexa. A second ultrasound scan will be performed on the sixth day of the cycle; if there is at least one follicle with a diameter of ≥12 mm, an ultrasound scan will be performed daily. When the dominant follicle's mean diameter is ≥16 mm, human chorionic gonadotropin - hCG (Ovitrelle® 250 μg; Merck, Kenilworth, NJ, USA) will be injected to trigger ovulation. Embryo transfer will be scheduled by the time of the hCG injection and embryo stages.

Serum progesterone level was also evaluated using the electrochemiluminescence immunoassay (Elecssys Progesterone III, Cobas®, Roche diagnosis, Germany) with a CV of 5.2%.

Serum progesterone was measured at 3 time points:

• 1st sample: On day 2 to day 4 of the cycle, before starting the endometrial preparation regime

• 2nd sample:

  • For AC protocol: Before administration of vaginal progesterone
  • For mNC protocol: Before hCG administration
  • For NC protocol: When an LH surge initiation was recorded, i.e., serum LH measured 20 IU/L or more

• 3rd sample: On the day of frozen embryo transfer at 8 a.m. Cycle cancellation

  • Artificial protocol: When the endometrial thickness is below 7mm after a duration of estradiol administration of ≥21 days or the emergence of a dominant follicle.
  • Natural cycle protocol: When there is no development of follicle, or no dominant follicle (≥14 mm), or no onset of LH surge observed after a duration of ≥21 days or unexpected spontaneous ovulation appears.
  • modified Natural cycle protocol: When there is no developing follicle (>16mm) observed after a duration of ≥21 days or pre-hCG unexpected spontaneous ovulation appears.
  • Both protocols: When there is no embryo surviving after thawing.

Frozen embryo transfer:

A maximum of 2 day-3 and one day-5 embryos will be thawed on the day of embryo transfer, three days after the start of progesterone. Two hours after thawing, surviving embryos will be transferred into the uterus under ultrasound guidance using a soft uterine catheter (Gynétics®, Belgium).

A series of progesterone levels evaluation will be performed at three times: (1) at the start of the cycle, (2) Before the time the embryo transfer is scheduled, (3) On the day of embryo transfer.

The blood sample at the start of the cycle will be stored for further epigenetics analysis.

Future babies' health will also be performed separately.

Inclusion Criteria:

• Aged of 18 to 45
• Having menstrual cycle duration of 24 to 38 days
• Undergoing no more than 3 previous IVF/ICSI - FET cycles
• Planning a frozen-thawed embryo transfer
• Agreeing to have no more than 2 day 3 and 1 day 5 embryos transferred
• Not participating in another IVF study at the same time

Exclusion Criteria:

• Menopausal/ Anovulatory women
• Having contraindication for exogenous hormones administration: breast cancer, risks of venous thromboembolism
• Having embryos from in vitro Maturation or oocyte donation or PGT (pre-implantation genetics testings) cycles
• Having uterine abnormalities (e.g., adenomyosis, intrauterine adhesions, unicornuate/ bicornuate/ arcuate uterus; unremoved hydrosalpinx, endometrial polyp)