Amphiregulin Versus Non-Amphiregulin Supplementation to Maturation Culturing Medium in IVM.
Condition: PCOS; IVM
Study type: Interventional

Summary: 
      Clinical use of IVM was pioneered in the nineties, but has not yet become a realistic option
      for wide-scale practice, for several reasons. Fundamentally, despite recent progress in
      improving the implantation and the pregnancy rates using in-vitro matured oocytes, results of
      IVM remain lower than treatment cycles utilizing conventional ART. To improve the outcome of
      IVM cycles, this study focuses on improving in-vitro culture conditions.

      In-vitro maturation (IVM) of human oocytes obtained from minimally stimulated or unstimulated
      ovaries offers a more "patient friendly" treatment option than the conventional Assisted
      Reproductive Technology (ART) treatment with controlled ovarian hyperstimulation (COH).
      Typically, IVM will be offered to women with polycystic ovaries (PCO/PCOS), or to patients
      with an excellent ovarian reserve, i.e. a high antral follicle count. IVM treatment is
      characterized by minimal administration of FSH or hMG and NO hCG trigger. The IVM approach is
      less disruptive to patients' daily life through the reduced need for hormonal and ultrasound
      monitoring, avoids a range of minor and major complications, such as ovarian hyperstimulation
      syndrome, and aims to reduce the total cost of infertility treatment for the patient and for
      the health care budget.

      Human oocytes retrieved from small antral follicles are able to resume meiosis by undergoing
      germinal vesicle breakdown and extrusion of the first polar body, if oocytes have reached
      meiotic competence. These oocytes can be fertilized although only a proportion (less than
      50%) of them can develop further into viable embryos. It has been hypothesized that failure
      of embryonic development may, at least in part, be due to an immature oocyte cytoplasm. A
      novel human in vitro maturation (IVM) culture system (named CAPACITATION-IVM is being
      investigated, hereafter named "CAPA") using 1°) natural compounds known to influence cAMP
      levels within the cumulus-oocyte-complex and 2°) compounds that are crucial for the
      oocyte-cumulus cross-talk. Keeping cyclic AMP high after retrieval in the GV oocyte prevents
      the occurrence of nuclear maturation, enabling increased communication between the oocyte and
      the cumulus cells. This allows for the improvement in the synchronization of nuclear and
      cytoplasmic maturation processes in the oocyte, to the benefit of embryo quality.
    

Detailed description: 
      There are two types of patients can be distinguished in this study:

        -  Polycystic ovarian morphology + normal cycle length (up to 35 days). The majority of
           subjects in this group have non-syndromic polycystic ovaries and do not have PCOS. In
           these patients, AMH is only moderately elevated and cyclical follicular development
           occurs under the influence of FSH.

        -  PCOS - Polycystic ovarian morphology + oligomenorrhoea (menstrual periods occurring at
           intervals of greater than 35 days, with only four to nine periods in a year) or
           amenorrhea. In these patients, with often strongly elevated AMH levels and concomitant
           hyperandrogenemia, relative FSH resistance results in arrest of follicular growth at the
           small antral follicular state. Spontaneous selection of a dominant follicle does not
           occur or occurs rarely. A large proportion of follicles are atretic. An important
           proportion of these patients have a BMI higher than 25. These patients generally receive
           the same standard gonadotropin stimulation regimen as the patients of the previous
           group, but if the largest follicles have a diameter of less than 8 mm on the third day
           of stimulation, patients receive one or two supplementary days of gonadotropin
           stimulation. Moreover, these patients generally take two-three weeks of OCP before the
           start of the IVM cycle and start stimulation approximately on day five after OCP
           withdrawal. However, since these patients do not have endogenous FSH-driven recruitment
           of follicles, the start of stimulation is rather flexible.

        -  The use of OCP (oral contraceptive pill) before the IVM cycle is obligate. OCP (for
           example Microgynon 30 micrograms) will be administered daily, to clear the atretic
           follicles in the ovary. OCP will be given for between 14 and 21 days (this method allows
           for better programming the cycles and the workload in the embryology lab).

      Before the first IVM cycle (Screening visit): All subjects will undergo a pelvic ultrasound
      scan to evaluate suitability to undergo IVM treatment. Patients will undergo a blood test for
      serology (Hepatitis B, HIV, syphilis) and baseline hormonal profiling (LH, FSH, E2,
      progesterone, AMH, SHBG, Testosterone). Additional analysis of TSH, thyroperoxidase
      antibodies, prolactin. Any concomitant medication taken in the last 3 months prior to the IVM
      attempt should be notified.

      First IVM treatment cycle:

        -  First clinic visit:

             -  All subjects will contact the study nurse on cycle day (cd1) to initiate the first
                Capacitation culture cycle. If cd1 is during the weekend, subjects will contact the
                study nurse on Monday. The subject will attend the clinic on day 1, 2 or 3 of
                menstrual bleeding. An ultrasound scan is performed to rule out the existence of
                ovarian cysts and a blood sample is taken for hormonal assessment (HCG, FSH, LH,
                E2, progesterone). On the evening of cycle day 3 of the first clinic visit,
                flexible Gonadotrophin stimulation will be started to enhance follicular
                development. The first dose of HP-hMG 150 IU at 2 PM. Second dose next day of
                HP-hMG 150 IU at 2 PM. The subject will return for a pelvic ultrasound scan and a
                blood test (FSH, LH, E2, progesterone) after the two days of HP-hMG 150 IU
                stimulation (i.e. on the morning of day 6).

             -  To patients with OCP bleeding: On day one, two, three, or four of the withdrawal
                bleeding that occurs after discontinuing the OCP, the subject will attend the
                clinic to undergo a pelvic ultrasound scan and a blood sample is taken for hormonal
                assessment (FSH, LH, E2, progesterone, AMH, total serum testosterone, SHBG). On the
                day of the first clinic visit, flexible HP-hMG stimulation will be started to
                enhance follicular development, at a first daily dose of HP-hMG at 8 P.M. The
                subject will return for a pelvic ultrasound scan and a blood test (FSH, LH, E2,
                progesterone) after two days of HP hMG stimulation.

        -  Second clinic visit at day 6 (after 2 days of HP-hMG treatment):

             -  If the ultrasound scan shows the presence of a single dominant follicle larger than
                12 mm with the other follicles all less than 10 mm, and the oocyte retrieval (OR)
                will be scheduled two days later, between 8:00 and 9:00 h. It is important to keep
                the OR 42 - 46 hours after the last HP-hMG injection as a fixed timing. If the
                ultrasound scan shows a maximal follicular diameter of less than 8 mm, a final
                injection of HP-hMG will be administered at 2 P.M. and the oocyte retrieval (OR)
                will be scheduled two days later, between 8:00 and 9:00 h. It is important to keep
                the OR 42 - 46 hours after the last HP-hMG injection as a fixed timing.

             -  To patients with OCP bleeding: If the ultrasound scan shows a maximal follicular
                diameter of approx. 9 mm, a final injection of HP-hMG will be administered at 2
                P.M. and the oocyte retrieval (OR) will be scheduled two days later, between 8:00
                and 9:00 h (under general anesthetics - the exact time of OR should be discussed
                with operating nurses and anesthetist). It is important to keep the time of OR
                constant at 42 -46 hours after the last HP-hMG injection.

        -  Third visit - Oocyte retrieval: Oocyte retrieval (OR) will be scheduled between 8:00 and
           10:00 h (under general anesthetics). On the day of OR:

             -  An ultrasound scan of both ovaries and of endometrial lining is recorded.

             -  A blood sample will be obtained for hormonal profiling (LH, FSH, E2, progesterone).

        -  All cumulus-oocyte complexes (COC) retrieved from the patient are cultured in CAPA
           medium for 24 hours.

        -  Following CAPA culture, half of oocytes will be divided randomly (50/50) to the two
           maturation triggers medium.

             -  Group 1: Medicult IVM media + AREG+FSH+HSA+Insulin+Estradiol (AREG-TRIGGER group)

             -  Group 2: Medicult IVM media + FSH+GH+hCG+HSA (CONTROL-TRIGGER group) with no AREG
                added

        -  In vitro oocyte maturation:

             -  Evaluate and register COCs for cumulus mass (CM) and cumulus contact between oocyte
                and cumulus cells. Each time take photographs. Discard fully denuded, degenerated
                oocytes.

             -  Transfer half of the COCs (5-10 at a time) from the Capacitation culture dish to
                the "washing dish" containing "Maturation Medium 1 (AREG-TRIGGER medium)" by using
                an Eppendorf micropipette and wash them thoroughly (load pipette tips with 5µl,
                max. 10µl). Then transfer COCs to the "IVM dish" with "Maturation Medium 1
                (AREG-TRIGGER medium)".

             -  Transfer the second half of the COCs from the Capacitation culture dish to the
                "washing dish" containing "Maturation Medium 2 (CONTROL-TRIGGER medium)" and wash
                them thoroughly (load pipette tips with 5µl, max. 10µl). Then transfer COCs to the
                "IVM dish" with "Maturation Medium 2 (CONTROL-TRIGGER medium)".

             -  Keep COCs in pools of ~10.

             -  Label the dish appropriately and culture COCs 30-32 hours in an incubator.

        -  Evaluation of maturation (MII, GVBD, GV) will be done at 30 hours. Oocytes which have
           undergone GVBD but with no clear PB will be assessed at 32 hours. Insemination will be
           performed using intra-cytoplasmic sperm injection (3-4 hours after oocyte retrieval or
           maturation check); only matured oocytes will be inseminated.

        -  Fertilization check will be performed under an inverted microscope at 16-18 hours after
           insemination. Embryo evaluation will be performed at 68 ±1 hours after fertilization
           using the Istanbul consensus. Standard Embryo Vitrification protocol used. Embryos will
           be vitrified per stimulation protocol obtained (group 1 AREG-TRIGGER or group 2 -
           CONTROL-TRIGGER).

        -  Frozen embryo transfer: The patient will be randomized to receive embryo randomly from
           AREG-TRIGGER or CONTROL-TRIGGER. Where no embryo(s) from the randomized group were
           available, embryo(s) from the other group were transferred.

        -  The first pregnancy test was performed 14 days after embryo transfer; a positive
           pregnancy test was defined as serum beta hCG >5 mIU/mL

        -  Clinical pregnancy was defined as at least one gestational sac on ultrasound at 7 weeks'
           gestation with the detection of heartbeat activity.

        -  Ongoing pregnancy was defined as pregnancy with a detectable heart rate at ≥12 weeks'
           gestation after the completion of the first transfer.

        -  Live birth was defined as the birth of at least one newborn after 24 weeks' gestation
           exhibiting any sign of life (twins were a single count).

        -  After a first unsuccessful IVM cycle: The clinical and embryological data and results
           related to the first IVM cycle (cumulative fresh and frozen embryo cycles) will be
           discussed, to establish a more patient-tailored approach for an eventual second IVM
           cycle. The patient-tailored approach in the second IVM cycle might consist of
           modification of the management of the follicular phase.
    

Intervention name(s): AREG-TRIGGER; CONTROL-TRIGGER
Intervention type(s): Drug; Drug
Intervention description(s): Oocytes in AREG-TRIGGER medium will be mature in AREG + FSH + HSA + Insulin + Estradiol medium.
Per case (6mL) 5940 µL "Basal Medium"
60 µL "IVM MIX" Do not need to filtrate media.; Oocytes in CONTROL-TRIGGER medium will be mature in FSH + hCG + GH + HSA medium.
Per case (5 mL) 4.3 ml IVM Medicult Medium (Vial 2) 0.5 ml HSA (from stock 10% solution) 50µl FSH (from stock 7.5 IU/ml) 5µl hCG (from stock 100 IU/ml) 75 µl GH (from stock 0.66mg/ml)
Other intervention name(s): Capacitation IVM medium; CAPA Control Medium
Allocation: Non-Randomized
Intervention model: Parallel Assignment
Masking: None (Open Label)
Primary purpose: Treatment