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Does the HCG trigger dose used for IVF impact luteal progesterone concentrations? a randomized controlled trial

  • Louise Svenstrup
    Correspondence
    Corresponding author.
    Affiliations
    Faculty of Health Sciences, Department of Clinical Research, University of Southern Denmark, Odense, Denmark

    Fertility Clinic, Unit of Gynecology and Obstetrics, Odense University Hospital, Odense, Denmark

    Research Unit of Gynecology and Obstetrics, Odense University Hospital, Odense, Denmark
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  • Sören Möller
    Affiliations
    Faculty of Health Sciences, Department of Clinical Research, University of Southern Denmark, Odense, Denmark

    OPEN, Odense Patient Data Explorative Network, Odense University Hospital, Odense, Denmark
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  • Jens Fedder
    Affiliations
    Faculty of Health Sciences, Department of Clinical Research, University of Southern Denmark, Odense, Denmark

    Fertility Clinic, Unit of Gynecology and Obstetrics, Odense University Hospital, Odense, Denmark

    Research Unit of Gynecology and Obstetrics, Odense University Hospital, Odense, Denmark
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  • Dorrit Elschner Pedersen
    Affiliations
    Fertility Clinic, Unit of Gynecology and Obstetrics, Odense University Hospital, Odense, Denmark
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  • Karin Erb
    Affiliations
    Fertility Clinic, Unit of Gynecology and Obstetrics, Odense University Hospital, Odense, Denmark
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  • Claus Yding Andersen
    Affiliations
    Laboratory of Reproductive Biology, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, Copenhagen, Denmark

    Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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  • Peter Humaidan
    Affiliations
    Faculty of Health Sciences, Department of Clinical Research, University of Southern Denmark, Odense, Denmark

    The Fertility Clinic, Skive Regional Hospital, Skive, Denmark

    Faculty of Health, Institute for Clinical Medicine, Aarhus, Aarhus University Hospital, Aarhus, Denmark
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      Abstract

      Research question

      Is there an association between the ovulation trigger dose of human chorionic gonadotrophin (HCG) and endogenous progesterone production during the luteal phase?

      Design

      This randomized controlled four-arm study, at the Fertility Clinic, Odense University Hospital, Denmark, included women undergoing gonadotrophin-releasing hormone (GnRH) antagonist IVF treatment with ≤11 follicles ≥12 mm. Group 1–3 were triggered with 5000 IU, 6500 IU or 10,000 IU HCG, respectively, receiving 17α-hydroxyprogesterone caproate intramuscularly for luteal-phase support (LPS) to measure endogenous progesterone production. Group 4 received 6500 IU HCG trigger and vaginal progesterone. During the study, the 5000 IU and 10,000 IU HCG groups were switched from urinary to recombinant HCG, as urinary HCG was removed from market. Eight blood samples were drawn during the luteal phase.

      Results

      Ninety-four participants completed the study. There was a significant positive association between the HCG trigger dose and the progesterone at 8 days (P < 0.001), 10 days (P < 0.001) and 14 days (P < 0.001) post-oocyte retrieval. Comparing the groups individually revealed a significant difference in progesterone concentration between low and high trigger doses at 4 days (P = 0.037) and 8 days (P = 0.007) post-oocyte retrieval and between all intervention groups at oocyte retrieval + 6 days: group 1 and 2 (P = 0.011), group 2 and 3 (P = 0.042) and group 1 and 3 (P < 0.001). Higher HCG trigger dose increased the progesterone from the individual follicle.

      Conclusions

      Increasing HCG trigger doses significantly increased endogenous progesterone concentration during the mid–late luteal phase.

      Keywords

      Introduction

      During the natural menstrual cycle, an LH surge around 40–60 IU/l induces ovulation and converts the Graafian follicle into a corpus luteum (
      • Andersen C.Y.
      • Kelsey T.
      • Mamsen L.S.
      • Vuong L.N.
      Shortcomings of an unphysiological triggering of oocyte maturation using human chorionic gonadotropin.
      ;
      • Roos J.
      • Johnson S.
      • Weddell S.
      • Godehardt E.
      • Schiffner J.
      • Freundl G.
      • Gnoth C.
      Monitoring the menstrual cycle: Comparison of urinary and serum reproductive hormones referenced to true ovulation.
      ). Subsequently, 4–10 IU/l of LH will support corpus luteum function and stimulate progesterone production during the early luteal phase. Progesterone peaks at around implantation 8 days after ovulation triggered by the natural LH surge, and a progesterone concentration ≥25 nmol/l during the natural cycle mid-luteal phase is considered to confirm ovulation and a well-functioning corpus luteum (
      • Reed B.G.
      • Carr B.R
      The Normal Menstrual Cycle and the Control of Ovulation.
      ;
      • Yding Andersen C.
      • Vilbour Andersen K.
      Improving the luteal phase after ovarian stimulation: reviewing new options.
      ). If pregnancy occurs, trophoblast-produced human chorionic gonadotrophin (HCG) will maintain the function of the corpus luteum until the luteo-placental shift, at around gestational week 7 (
      • Fatemi H.M.
      The luteal phase after 3 decades of IVF: what do we know?.
      ;
      • Järvelä I.Y.
      • Ruokonen A.
      • Tekay A.
      Effect of rising hCG levels on the human corpus luteum during early pregnancy.
      ).
      In assisted reproductive technology (ART), the luteal phase is abnormal due to supra-physiological steroid concentrations produced by several corpora lutea as a result of ovarian stimulation with exogenous gonadotrophins. The increased steroid concentration reduces the endogenous pituitary release of LH and FSH via negative feedback on the hypothalamic–pituitary axis, which, if unsupported, will result in the demise of the corpus luteum (
      • Edwards R.G.
      • Steptoe P.C.
      • Purdy J.M.
      Establishing full-term human pregnancies using cleaving embryos grown in vitro.
      ;
      • Fatemi H.M.
      • Popovic-Todorovic B.
      • Papanikolaou E.
      • Donoso P.
      • Devroey P.
      An update of luteal phase support in stimulated IVF cycles.
      ;
      • Fauser B.C.
      • Devroey P.
      Reproductive biology and IVF: ovarian stimulation and luteal phase consequences.
      ).
      HCG is used as a surrogate for the endogenous LH to secure final oocyte maturation in ART (
      • Andersen C.Y.
      • Kelsey T.
      • Mamsen L.S.
      • Vuong L.N.
      Shortcomings of an unphysiological triggering of oocyte maturation using human chorionic gonadotropin.
      ). HCG and LH are similar in structure and bind to the same receptor; however, HCG has a significantly longer half-life than LH and is currently available in a urinary as well as a recombinant form with similar pharmacokinetic and pharmacodynamic profiles (
      • Trinchard-Lugan I.
      • Khan A.
      • Porchet H.C.
      • Munafo A.
      Pharmacokinetics and pharmacodynamics of recombinant human chorionic gonadotrophin in healthy male and female volunteers.
      ).
      After a 6500 IU recombinant HCG (rHCG) trigger, HCG reaches its peak in the serum at 130 IU after 24 h and returns to its pre-trigger baseline concentration at oocyte retrieval + 6 days (day 8 of the trigger;
      • Vuong L.N.
      • Ho T.M.
      • Pham T.D.
      • Ho V.N.A.
      • Andersen C.Y.
      • Humaidan P.
      The early luteal hormonal profile in IVF patients triggered with hCG.
      ). From this day onwards, the corpus luteum depends on endogenous LH production which, as mentioned above, is suppressed due to supra-physiological circulating steroid concentrations (
      • Beckers N.G.
      • Macklon N.S.
      • Eijkemans M.J.
      • Ludwig M.
      • Felberbaum R.E.
      • Diedrich K.
      • Bustion S.
      • Loumaye E.
      • Fauser B.C.
      Nonsupplemented luteal phase characteristics after the administration of recombinant human chorionic gonadotropin, recombinant luteinizing hormone, or gonadotropin-releasing hormone (GnRH) agonist to induce final oocyte maturation in in vitro fertilization patients after ovarian stimulation with recombinant follicle-stimulating hormone and GnRH antagonist cotreatment.
      ;
      • Weissman A.
      • Lurie S.
      • Zalel Y.
      • Goldchmit R.
      • Shoham Z.
      Human chorionic gonadotropin: pharmacokinetics of subcutaneous administration.
      ). Although the implanted embryo secretes detectable concentrations of HCG from approximately 8 days after oocyte retrieval, a period of deficiency of LH activity occurs during the peri-implantation phase; thus, exogenous luteal phase support (LPS) is mandatory until the early trophoblast secretes sufficient amounts of HCG to rescue the corpus luteum (
      • Beckers N.G.
      • Macklon N.S.
      • Eijkemans M.J.
      • Ludwig M.
      • Felberbaum R.E.
      • Diedrich K.
      • Bustion S.
      • Loumaye E.
      • Fauser B.C.
      Nonsupplemented luteal phase characteristics after the administration of recombinant human chorionic gonadotropin, recombinant luteinizing hormone, or gonadotropin-releasing hormone (GnRH) agonist to induce final oocyte maturation in in vitro fertilization patients after ovarian stimulation with recombinant follicle-stimulating hormone and GnRH antagonist cotreatment.
      ;
      • de Ziegler D.
      • Pirtea P.
      • Andersen C.Y.
      • Ayoubi J.M.
      Role of gonadotropin-releasing hormone agonists, human chorionic gonadotropin (hCG), progesterone, and estrogen in luteal phase support after hCG triggering, and when in pregnancy hormonal support can be stopped.
      ;
      • Fatemi H.M.
      • Popovic-Todorovic B.
      • Papanikolaou E.
      • Donoso P.
      • Devroey P.
      An update of luteal phase support in stimulated IVF cycles.
      ;
      • Humaidan P.
      • Bredkjaer H.E.
      • Bungum L.
      • Bungum M.
      • Grøndahl M.L.
      • Westergaard L.
      • Andersen C.Y.
      GnRH agonist (buserelin) or hCG for ovulation induction in GnRH antagonist IVF/ICSI cycles: a prospective randomized study.
      ).
      Endogenous progesterone has several important functions during the luteal phase apart from supporting early implantation. Progesterone regulates the window of implantation via secretory transformation of the endometrium, increases vascularity of the endometrium, functions as an immune modulator, and reduces uterine contractions peri-implantation (
      • Fatemi H.M.
      • Popovic-Todorovic B.
      • Papanikolaou E.
      • Donoso P.
      • Devroey P.
      An update of luteal phase support in stimulated IVF cycles.
      ;
      • Yding Andersen C.
      • Vilbour Andersen K.
      Improving the luteal phase after ovarian stimulation: reviewing new options.
      ).
      Regarding the association between progesterone concentration and live birth in fresh transfer IVF cycles, a recent study reported an optimal concentration of 60–100 nmol/l during the early luteal phase, 2–3 days after oocyte retrieval, and 150–250 nmol/l (approximately 50–80 ng/ml) during the mid-luteal phase, 5 days after oocyte retrieval (
      • Thomsen L.H.
      • Kesmodel U.S.
      • Andersen C.Y.
      • Humaidan P.
      Daytime Variation in Serum Progesterone During the Mid-Luteal Phase in Women Undergoing In Vitro Fertilization Treatment.
      ). In cryopreservation cycles with hormone replacement therapy a low cut-off concentration of progesterone has been suggested to be approximately 35 nmol/l (10 ng/ml;
      • Alsbjerg B.
      • Thomsen L.
      • Elbaek H.O.
      • Laursen R.
      • Povlsen B.B.
      • Haahr T.
      • Humaidan P.
      Progesterone levels on pregnancy test day after hormone replacement therapy-cryopreserved embryo transfer cycles and related reproductive outcomes.
      ;
      • Melo P.
      • Chung Y.
      • Pickering O.
      • Price M.J.
      • Fishel S.
      • Khairy M.
      • Kingsland C.
      • Lowe P.
      • Petsas G.
      • Rajkhowa M.
      • Sephton V.
      • Tozer A.
      • Wood S.
      • Labarta E.
      • Wilcox M.
      • Devall A.
      • Gallos I.
      • Coomarasamy A.
      Serum luteal phase progesterone in women undergoing frozen embryo transfer in assisted conception: a systematic review and meta-analysis.
      ). However, the issue of the correlation between serum progesterone measurement and endometrial receptivity in FET cycles has recently been debated (
      • Lawrenz B.
      • Fatemi H.M.
      Are serum progesterone measurements truly representative for the identification of an adequate luteal phase in hormonal replacement therapy frozen embryo transfers?.
      ). Importantly, in fresh transfer IVF cycles the contribution of the exogenously administered progesterone to the serum progesterone concentration is relatively modest, at approximately 30–40 nmol/l (9.5 ng/ml) when the vaginal route is used, and there seems to be an upper level for vaginal absorption (
      • Alsbjerg B.
      • Thomsen L.
      • Elbaek H.O.
      • Laursen R.
      • Povlsen B.B.
      • Haahr T.
      • Humaidan P.
      Progesterone levels on pregnancy test day after hormone replacement therapy-cryopreserved embryo transfer cycles and related reproductive outcomes.
      ;
      • Levine H.
      • Watson N.
      Comparison of the pharmacokinetics of crinone 8% administered vaginally versus Prometrium administered orally in postmenopausal women(3).
      ).
      Varying doses of HCG, either 5000 IU, 6500 IU or 10,000 IU, have randomly been used to trigger final oocyte maturation, as dosing below 5000 IU was previously shown to decrease the number of oocytes retrieved (
      • Abdalla H.I.
      • Ah-Moye M.
      • Brinsden P.
      • Howe D.L.
      • Okonofua F.
      • Craft I.
      The effect of the dose of human chorionic gonadotropin and the type of gonadotropin stimulation on oocyte recovery rates in an in vitro fertilization program.
      ). The standard HCG trigger dose used by the majority of IVF doctors is 6500 IU (
      • Tobler K.J.
      • Zhao Y.
      • Weissman A.
      • Majumdar A.
      • Leong M.
      • Shoham Z.
      Worldwide survey of IVF practices: trigger, retrieval and embryo transfer techniques.
      ). Even though a 5000 IU HCG trigger is sufficient to obtain final oocyte maturation, results from studies using different trigger doses and their effect on circulating progesterone luteal concentrations are ambiguous (
      • Chan C.C.
      • Ng E.H.
      • Tang O.S.
      • Yeung W.S.
      • Lau E.Y.
      • Ho P.C.
      A prospective, randomized, double-blind study to compare two doses of recombinant human chorionic gonadotropin in inducing final oocyte maturity and the hormonal profile during the luteal phase.
      ;
      • Driscoll G.L.
      • Tyler J.P.
      • Hangan J.T.
      • Fisher P.R.
      • Birdsall M.A.
      • Knight D.C.
      A prospective, randomized, controlled, double-blind, double-dummy comparison of recombinant and urinary HCG for inducing oocyte maturation and follicular luteinization in ovarian stimulation.
      ; International Recombinant Human Chorionic Gonadotropin Study
      Group, I.R.H.C.G.S.
      Induction of ovulation in World Health Organization group II anovulatory women undergoing follicular stimulation with recombinant human follicle-stimulating hormone: a comparison of recombinant human chorionic gonadotropin (rhCG) and urinary hCG.
      ;
      • Kovacs P.
      • Kovats T.
      • Bernard A.
      • Zadori J.
      • Szmatona G.
      • Kaali S.G.
      Comparison of serum and follicular fluid hormone levels with recombinant and urinary human chorionic gonadotropin during in vitro fertilization.
      ). However, the results of those studies were based on measurements of total serum progesterone concentration and did not distinguish the contribution of the exogenous progesterone from the endogenous progesterone production driven by the HCG trigger.
      The primary aim of this randomized study was to investigate the impact of different HCG trigger doses on endogenous progesterone production from the corpus luteum, using 17α-hydroxyprogesterone caproate for LPS to distinguish the endogenous progesterone production from the exogenous supplementation. Secondary aims were to explore the association between the number of follicles and oocytes retrieved per patient and the luteal progesterone concentration; finally, the authors wanted to investigate the progesterone production from each follicle ≥12 mm and ≥14 mm to explore whether increasing the HCG trigger dose would increase progesterone production from the individual corpora lutea.

      Materials and methods

      Design

      This was a randomized controlled multi-arm trial conducted at the Fertility Clinic of Odense University Hospital between January 2015 and September 2019.

      Study population

      Women aged 18–40 years who were undergoing gonadotrophin-releasing hormone (GnRH) antagonist co-treated IVF, had normal baseline FSH concentrations ≤12 IU/l, LH concentrations ≤15 IU/l and oestradiol concentrations, thyroid and prolactin hormone concentrations within the normal ranges were invited to participate. Each woman could participate only once. The exclusion criteria were body mass index (BMI) <18 kg/m2 or >35 kg/m2, clinically significant disease of the heart, liver or kidneys, epilepsy, endocrine diseases such as diabetes or metabolic disorders, coagulation disorders, including a previous embolus or thrombus, HIV or hepatitis. Moreover, patients were excluded if embryo transfer was cancelled. Finally, participants who developed ≥12 follicles measuring ≥12 mm on the last day of ultrasonography before the trigger or who were assessed to be at high risk of developing ovarian hyperstimulation syndrome (OHSS) were excluded.

      Ovarian stimulation, laboratory procedures and embryo scoring

      Participants received oral and written information at the initiation of ovarian stimulation. The stimulation protocol was individualized, based on antral follicle count, age, BMI and, when available, the response to previous ovarian stimulation. Ovarian stimulation started on cycle day 2 or 3 using recombinant FSH (rFSH; Gonal-f, Merck, Denmark; Rekovelle, Ferring, Denmark; or Puregon, Organon, Denmark), urinary FSH (Fostimon, IBSA Nordic, Denmark), corifollitropin-alfa (Elonva, MSD, Denmark), rFSH/LH (Pergoveris, Merck, Denmark) or human menopausal gonadotrophin (HMG; Menopur, Ferring, Denmark) alone or in combination. Ultrasonography was performed regularly during the follicular phase and the gonadotrophin dose was adjusted if necessary. GnRH antagonist co-treatment was started when the leading follicle reached a size of 12 mm. As soon as three follicles reached a diameter of 18 mm, oocyte retrieval was planned 36 h after the ovulation trigger with either 5000 IU, 6500 IU or 10,000 IU HCG. Laboratory procedures were similar for all patients and in accordance with the unit's standard practice. Embryos and blastocysts were scored using Gardner's score (
      • Gardner D.K.
      • Lane M.
      • Stevens J.
      • Schlenker T.
      • Schoolcraft W.B.
      Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer.
      ). Generally, one or a maximum of two cleavage-stage embryos or one blastocyst were transferred on day 2 or 5, respectively.

      Ultrasound assessment

      During the last vaginal ultrasound scan before the ovulation trigger, all follicles measuring ≥12 mm in each ovary were recorded. Follicles were measured in two dimensions and the average measurement was recorded.

      Randomization

      On the day of the final ultrasound scan, usually on the ovulation trigger day or the day before, participants were randomized into one of four groups. Randomization was computerized via the website www.random.org using a random sequence generator. Doctors enrolled the participants, and doctors or nurses assigned them to the intervention. Block randomization was used to secure an equal allocation ratio among the groups. The study was unblinded. For every woman excluded, one extra patient was included with a new participant and randomization number.

      HCG trigger, LPS and compliance

      A total of 127 patients were randomized into three study groups and a control group, giving a total of four groups. Group 1 received ovulation triggering with 5000 IU urinary HCG (uHCG; Pregnyl, Organon, Denmark). Group 2 had ovulation triggering with 6500 IU rHCG (Ovitrelle; Merck, Denmark). Group 3 underwent ovulation triggering with 10,000 IU uHCG (Pregnyl). In Groups 1, 2 and 3, LPS consisted of 341 mg 17α-hydroxyprogesterone caproate (Lentogest, IBSA Nordic, Denmark) administered intramuscularly every third day. Patients in these three groups had a total of five luteal phase injections. The women were instructed and trained to undertake the injection themselves, into their femoral muscle or alternatively their gluteus muscle.
      Group 4, the control group, had triggering with 6500 IU rHCG, with standard LPS consisting of a total of 180 mg micronized progesterone that was administered vaginally in two separate daily doses (Crinone, Merck, Denmark).
      Doses of 5000 IU uHCG, 10,000 IU uHCG and 6500 IU of rHCG were chosen as they were the standard HCG trigger doses used in the daily clinical setting at the time of the study planning. The medication of the control groups was in line with the standard approach for poor and normal responders in the clinic.
      During the study period, in March 2018, uHCG (Pregnyl) was discontinued. Following this date, instead of 5000 and 10,000 IU uHCG (Pregnyl), group 1 received 4940 IU rHCG (Ovitrelle) and group 3 received 10,140 IU rHCG (Ovitrelle). Ovitrelle rHCG comes as a prefilled pen, and the lowest dose that can be administrated is 260 IU rHCG. The doses were selected because they were close to the primary doses. Overall, 81 of the 127 patients (64%) were randomized before uHCG (Pregnyl) was discontinued.
      In all groups, LPS started the day after oocyte retrieval and continued until the day of the pregnancy test – the day of oocyte retrieval + 14 days. Patient compliance was monitored by a personal completed participant form and by the return of all empty medicine boxes.

      Blood sampling and hormone assays

      A total of eight blood samples were drawn from each participant: on the day of the last ultrasound scan (usually on the day of the trigger or the day before), on the day of oocyte retrieval, then 2, 4, 6, 8 and 10 days subsequently, and also on the day of the pregnancy test, oocyte retrieval + 14 days. Blood samples were collected independently of the embryo transfer day. Three 10 ml uncoated tubes of full blood were taken at each of the eight visits. After coagulation at room temperature, the full blood was centrifuged at 400g for 15 min. Serum was isolated and frozen at –20°C and subsequently at –80°C.
      Progesterone and 17α-hydroxyprogesterone were assessed at the Department of Clinical Biochemistry and Pharmacology of Odense University Hospital using an automated electro-chemiluminescent immunoassay (Immunite 2000 XPi, Siemens Healthcare, Denmark) and a TSQ Quantiva TLX-2 high-performance liquid chromatography system (Thermo Scientific, USA) respectively.
      The calibration and measurement range for progesterone was 0.64–127 nmol/l; values over the limit were diluted and reanalysed to obtain exact values. In the laboratory the intermediary precision was 4.4% and inter-serial precision was 1.6%. For 17α-hydroxyprogesterone the measurement range was 0.2–125 nmol/l, the intermediary precision 6.8%, and the inter-serial precision 4.9%.
      Blood samples were analysed for progesterone from all eight visits and were analysed for 17α-hydroxyprogesterone at visits at oocyte retrieval + 6 days and retrieval + 8 days during the mid-luteal phase.

      Reproductive outcome

      Pregnancy was defined as a serum β-HCG concentration of ≥50 IU/l 14 days after oocyte retrieval. Elevated serum β-HCG (5–49 IU/L) was controlled until a negative or positive value. A clinical pregnancy was defined as the presence of an intrauterine fetus with a heartbeat in gestational week 7–8. Early missed and spontaneous abortions were defined as a pregnancy without a living fetus at week 7–8. Participants were followed until a negative pregnancy test was obtained or a clinical pregnancy was confirmed.

      Ethics

      This study was approved by the local Ethics Committee of Southern of Denmark (registration number S-20130161, 22.05.2014) and the Danish Health and Medicines Authority (registration number 13.010, 16.07.2014) and registered with the Danish Data Protection Agency (registration number 14/23490, 12.06.2014). The study was monitored by the local Good Clinical Practice Unit and conducted according to the Declaration of Helsinki. All patients gave written informed consent. The study was also registered with the European Community Clinical Trail System (EudraCT; registration number 2013-003304-39). The study is reported according to the CONSORT statement.

      Sample size calculation

      As no previous studies had explored the endogenous progesterone concentration after varying HCG trigger doses, it was not possible, when the study was planned, to predict the expected difference in progesterone. As a result, the power calculation was based on the variance ratio, a type II error of 20% and the fact that the study involved four groups. In total 92, participants were needed to provide a 80% power to detect a significant level of 5% (P < 0.05) in progesterone concentration between the groups.
      The power calculation was based on a one-way analysis of variance analysis, but once the study was finalized, it was decided to use the mixed-effects model to use the longitudinal design with repeated measurements over time.

      Statistics

      Normality was assessed using the Shapiro–Wilk test. Normally distributed continuous data were analysed with linear regression using an F-test and were expressed as means and standard deviations. The median test was used for continuous data without a normal distribution, and data were presented as medians and percentiles. Categorical data were analysed by Fisher's exact test and expressed as counts and proportions. Mixed-effects linear regression models were used to test differences in the primary outcome between the groups. Furthermore, each group was individually analysed against each of the three other groups. Data were adjusted for pregnancy, follicle count and number of retrieved oocytes. A P-value <0.05 was considered statistically significant. All analyses were carried out using STATA (StataCorp. 2019. College Station, TX: StataCorp LLC, USA) version 16.1.

      Results

      Randomization

      Overall, a total of 127 women were randomized to the study, and 94 (74%) completed it. Figure 1 presents the flow chart of patient allocation. A total of 33 (26%) patients were secondarily excluded due to a cancellation of embryo transfer caused by either a lack of oocytes at retrieval, a lack of fertilization, poor development of the embryo or missing blood samples. The total numbers of participants with analysed blood samples in each group were 21, 22, 25 and 26, respectively. There were no significant differences in the number of women excluded between the groups (P = 0.486).
      Figure 1
      Figure 1Flow chart of study participation, inclusion, exclusion and randomization.

      Patient characteristics

      The baseline characteristics of the participants are shown in Table 1. The four groups were similar as regards age, BMI, FSH concentration, smoking status, alcohol intake, previous pregnancies and deliveries, number of previous intrauterine insemination and IVF treatments, and duration of infertility. LH concentration and cycle length were the only baseline characteristics that differed between the groups. The cycle length was longer in group 3 as one of the patients had severe oligomenorrhoea. Regarding the distribution of the diagnoses, the primary cause of infertility was similar between the four groups. Idiopathic and male infertility were the most frequent causes of infertility, with an overall prevalence in the groups that ranged from 62% to 91% (Supplementary Table 1).
      Table 1Baseline patient characteristics for the four treatment groups
      Parameter5000 IU HCG + 17α- hydroxyprogesterone (n = 21)6500 IU HCG + 17α hydroxyprogesterone (n = 22)10,000 IU HCG + 17α- hydroxyprogesterone OH P4 (n = 25)6500 IU HCG + progesterone (n = 26)
      Age (years), mean (SD)29.1 (5.0)31.1 (4.44)30.1 (4.4)31.7 (4.3)
      Duration of infertility (years), median (25th and 75th percentiles)2.0 (1.0; 3.0)2.0 (2.0; 3.0)2.0 (1.0; 3.0)2.0 (2.0; 3.0)
      Cycle length (days), median (25th and 75th percentiles)27.0 (26.0; 28.0)28.5 (28.0; 30.0)30.0 (28.0; 32.0)28.0 (27.0; 29.0)
      Previous pregnancy, median (25th and 75th percentiles)1.0 (0.0; 1.0)1.0 (0.0; 1.0)0.0 (0.0; 0.0)0.0 (0.0; 1.0)
      Previous delivery,

      median (25th and 75th percentiles)
      0.0 (0.0; 0.0)0.0 (0.0; 0.0)0.0 (0.0; 0.0)0.0 (0.0; 0.0)
      LH (IU/I), mean (SD)4.4 (2.0)5.2 (2.2)6.9 (2.4)4.9 (2.4)
      BMI (kg/m2), median (25th and 75th percentiles)25.2

      (24.6; 28.9)
      24.3

      (22.3; 28.3)
      25.0

      (21.4; 30.1)
      25.4

      (23.0; 27.1)
      Smoking,

      n (%)
      3 (14)3 (14)2 (8)2(8)
      Alcohol,

      n (%)
      0 (0)3 (14)3 (12)2 (8)
      HCG, human chorionic gonadotrophin.

      Ovarian stimulation

      There were no differences between the four groups as regards total FSH/HMG consumption, total number of days of stimulation, and endometrial thickness on the day of the last ultrasound scan. Furthermore, no difference was seen in terms of the number of follicles measuring ≥12 mm and ≥14 mm. The median number of follicles ≥12 mm and ≥14 mm at the last vaginal ultrasonography before ovulation triggering was 8.5 and 6.0, respectively. Participants underwent stimulation for a median of 10.0 days, and the median oocyte retrieval day was cycle day 13.0. Overall, 70 (74%) of women were stimulated using FSH and 24 (26%) using a combination of FSH and LH, with no difference in outcomes seen between the groups (Table 2).
      Table 2Ovarian stimulation characteristics of the four treatment groups
      Parameter5000 IU HCG + 17α- hydroxyprogesterone (n = 21)6500 IU HCG + 17α hydroxyprogesterone (n = 22)10,000 IU HCG + 17α- hydroxyprogesterone OH P4 (n = 25)6500 IU HCG + progesterone (n = 26)P-value
      Total FSH/HMG dose (IU)2025.0 (1600.0; 2700.0)1975.0 (1000.0; 2250)1637.5 (1175.0; 2250.0)1912.5 (1575.0; 2300.0)0.899
      Women receiving stimulation containing LH4 (19)3 (14)8 (32)9 (35)0.310
      Days of stimulation10.0 (9.0; 13.0)10.0 (8.0; 11.0)10.0 (9.0; 12.0)10.0 (9.0; 11.0)0.698
      Number of follicles ≥12 mm8.0 (6.0; 12.0)9.0 (6.0; 10.0)9.0 (6.0; 11.0)6.5 (5.0; 9.0)0.698
      Number of follicles ≥14 mm6.0 (4.0; 7.0)6.0 (4.0; 8.0)6.0 (4.0; 8.0)5.0 (4.0; 8.0)0.918
      Endometrial thickness (mm)9.6 (8.2; 11.5)9.1 (7.7; 10.0)9.3 (8.3; 10.7)9.3 (7.1; 10.7)0.676
      Transfer0.659
      Embryo15 (72)

      18 (82)17 (68)20 (77)
      Blastocyst6 (29)4 (18)8 (32)6 (23)
      Data are n (%) or median (25th and 75th percentiles).
      HCG, human chorionic gonadotrophin.

      Oocytes and embryos

      No differences were seen in the number of follicles aspirated and the number of retrieved, fertilized and cleaved oocytes between the groups. The median number of oocytes retrieved was 6.6, ranging from 1.0 to 15.0 oocytes. The number of normally fertilized and divided oocytes was a median of 4.0 per patient. In the majority of cycles, homologous spermatozoa were used for insemination, and the most frequent fertilization method was IVF. The four groups were similar as regards fertilization method and sperm source. For most of the participants (73%) an embryo was transferred 2 days after oocyte retrieval, with one having an embryo transfer on day 3; 26% of women had a blastocyst transfer, and there was no difference between the groups (P = 0.659). The median number of additional blastocysts to be cryopreserved was 1.0, with no differences between the groups (Supplementary Table 2). Only one patient underwent the transfer of two embryos, and this patient did not conceive.

      Serum progesterone

      Figure 2a presents an overview of the median progesterone value in each group. In all groups, progesterone was low on the last day of ultrasonography, before the ovulation trigger. The highest progesterone value was 7.4 nmol/l and only three women had a basal progesterone ≥5 nmol/l before ovulation triggering. At oocyte retrieval, 36 h after the ovulation trigger, the progesterone values were marginally increased and relatively equal between the groups. Subsequently, progesterone concentration increased in all the groups until oocyte retrieval + 4 days, reaching a peak concentration at oocyte retrieval + 4 days. There were no significant differences between the groups on the last day of ultrasonography (P = 0.274), oocyte retrieval (P = 0.905) or 2 (P = 0.696), 4 (P = 0.147) and 6 (P = 0.227) days post-retrieval.
      Figure 2
      Figure 2Serum concentrations of progesterone at randomization, at oocyte retrieval and at 2, 4, 6 8, 10 and 14 days post-retrieval. (A) Median progesterone concentrations for the participants in each group. (B) Median progesterone concentrations for pregnant women in each group. (C) Median progesterone concentrations for non-pregnant women in each group. Medical intervention in the groups: group 1: 5000 IU HCG + 17α-hydroxyprogesterone; group 2: 6500 IU HCG + 17α-hydroxyprogesterone; group 3: 10.000 IU HCG + 17α-hydroxyprogesterone; group 4: 6500 IU HCG + progesterone. HCG, human chorionic gonadotrophin; US, ultrasonography.
      The progesterone concentration rapidly decreased from oocyte retrieval + 4 days in group 1, and oocyte retrieval + 6 days in group 2, 3 and 4. There were significant differences between the four groups on days 8 (P < 0.001), 10 (P < 0.001) and 14 (P < 0.001) post-oocyte retrieval (Table 3).
      Table 3Progesterone and 17α-hydroxyprogesterone concentrations for the four treatment groups
      Parameter5000 IU HCG + 17α-hydroxyprogesterone (n = 21)6500 IU HCG + 17α hydroxyprogesterone (n = 22)10,000 IU HCG + 17α- hydroxyprogesterone (n = 25)6500 IU HCG + progesterone (n = 26)P-value
      Progesterone (nmol/l), median (25th and 75th percentiles)
      Day of last ultrasonography

      (n = 92)
      1.9 (1.5; 2.3)2.1 (1.3; 2.9)1.6 (1.0; 2.4)2.3 (1.6; 2.7)0.274
      Day of oocyte retrieval

      (n = 94)
      20.4 (15.7; 28)21.4 (17.7; 35.0)16.8 (11.1; 25.7)21.0 (17.3; 31.0)0.905
      Retrieval + 2 days

      (n = 93)
      79.5 (60.7; 110.0)104.0 (74.4; 168.0)94.1 (58.2; 177.0)125.5 (70.0; 246.0)0.696
      Retrieval + 4 days

      (n = 91)
      123.5 (105.0; 281.0)228 (104.0; 287.0)234.5 (120.0; 364.0)286.5 (125.0; 382.0)0.147
      Retrieval + 6 days

      (n = 93)
      61.7 (36.0; 181.0)171.5 (89.7; 285.0)216 (125.0; 299.0)207.0 (82.7; 278.0)0.227
      Retrieval + 8 days

      (n = 94)
      8.4 (6.4; 24.5)19.1 (13.0; 59.1)47.4 (31.4; 116.0)47.2 (33.1; 63.6)<0.001
      Retrieval +10 days

      (n = 91)
      3.8 (1.2; 7.4)5.8 (3.5; 10.4)11.7 (7.1; 56.0)36.1 (28.0; 45.8)<0.001
      Retrieval + 14 days

      (n = 89)
      1.6 (1.2; 7.4)1.5 (1.1; 4.4)2.6 (1.1; 90.3)34.0 (27.1; 48.0)<0.001
      17α-Hydroxyprogesterone (nmol/l), median (25th and 75th percentiles)
      Day of oocyte retrieval + 6 days

      (n = 93)
      9.6 (6.7; 30.7)26.0 (13.5; 42.9)36.8 (21.3; 60.5)22.9 (11.7; 36.7)0.069
      Retrieval + 8 days

      (n = 94)
      1.7 (1.2; 3.5)3.2 (1.9; 10.7)8.0 (5.3; 20.7)3.8 (2.0; 5.7)0.001
      HCG, human chorionic gonadotrophin.
      Progesterone concentrations compared between the groups individually revealed differences earlier during the mid-luteal phase. At oocyte retrieval + 4 days, a statistical difference was seen between group 1 and 3 (P = 0.037) and group 1 and 4 (P = 0.004). Close to the implantation window at oocyte retrieval + 6 days, statistical differences were detected between several of the groups individually: between group 1 and 2 (P = 0.011), group 1 and 3 (P < 0.001), group 1 and 4 (P < 0.001), and group 2 and 3 (P = 0.042). Significant differences were also seen at oocyte retrieval + 8 days between group 1 and 4 (P = 0.047) and group 1 and 3 (P = 0.007) (Table 4).
      Table 4Individually significant differences between the groups and adjustments for pregnancy, follicle size and oocytes retrieved
      TimeProgesterone (P-value)Adjusted for pregnancy (P-value)Adjusted for follicle count ≥12 mm (P-value)Adjusted for follicle count ≥14 mm (P-value)Adjusted for follicle count both ≥12 mm and ≥14 mm (P-value)Adjusted for number of mature oocytes retrieved (P-value)
      Last scanNSNSNSNSNSNS
      Oocyte retrievalNSNSNSNSNSNS
      Retrieval + 2 daysNSGroup 1–4 (0.049)Group 1–4 (0.022)NSGroup 1–4 (0.039)NS
      Retrieval + 4 daysGroup 1–3 (0.037)

      Group 1–4 (0.004)
      Group 1–4 (0.002)

      Group 1–3 (0.029)

      Group 2–4 (0.038)
      Group 1–4 (≤0.001)

      Group 1–2 (0.003)

      Group 1–3 (0.022)

      Group 2–4 (0.003)
      Group 1–4 (0.002)

      Group 2–4 (0.030)
      Group 1–4 (0.001)

      Group 1–3 (0.032)

      Group 2–4 (0.019)
      Group 1–4 (0.005)

      Group 1–3 (0.036)
      Retrieval + 6 daysGroup 1–2 (0.011)

      Group 1–3 (<0.001)

      Group 1–4 (<0.001)

      Group 2–3 (0.042)
      Group 1–2 (0.008)

      Group 1–3 (<0.001)

      Group 1–4 (<0.001)

      Group 2–3 (0.030)
      Group 1–4 (≤0.001)

      Group 2–4 (0.043)

      Group 1–2 (0.008)

      Group 1–3 (≤0.001)

      Group 2–3 (0.010)
      Group 1–2 (0.005)

      Group 1–3 (<0.001)

      Group 1–4 (<0.001)
      Group 1–2 (0.009)

      Group 1–3 (≤0.001)

      Group 1–4 (≤0.001)

      Group 2–3 (0.025)
      Group 1–2 (0.003)

      Group 1–3 (<0.001)

      Group 1–4 (<0.001)
      Retrieval + 8 daysGroup 1–3 (0.007)

      Group 1–4 (0.047)
      Group 1–3 (0.005)

      Group 1–4 (0.046)
      Group 1–3 (0.003)

      Group 1–4 (0.018)
      Group 1–3 (0.004)

      Group 1–4 (0.028)
      Group 1–3 (0.005)

      Group 1–4 (0.021)
      Group 1–3 (0.005)

      Group 1–4 (0.038)
      Retrieval + 10 daysNSNSNSNSNSNS
      Retrieval + 14 daysNSNSGroup 2–3 (0.042)

      Group 2–4 (0.035)
      Group 2–4 (0.045)Group 2–4 (0.043)NS
      NS, not significant.
      Furthermore, there were no differences in progesterone concentration between participants who received uHCG or rHCG in group 1 and 3, respectively.

      Serum 17α-hydroxyprogesterone

      In the four groups of participants, 17α-hydroxyprogesterone was highest at oocyte retrieval + 6 days compared with retrieval + 8 days. The individual group analyses of 17α-hydroxyprogesterone concentration showed a significant difference between several groups at oocyte retrieval + 6 days: group 1 and 2 (P = 0.027), group 1 and 3 (P < 0.001), group 2 and 3 (P = 0.001) and group 3 and 4 (P < 0.001). At oocyte retrieval + 8 days, significant differences were seen between group 1 and 3 (P = 0.002), group 2 and 3 (P = 0.040) and group 3 and 4 (P = 0.012) (data not shown).

      Reproductive outcome

      Of a total of 30 (32%) women who had a positive pregnancy test, 22 (23%) had a clinical pregnancy. After a positive pregnancy test, eight participants had an early missed or spontaneous abortion. No significant difference in pregnancy or clinical pregnancy rates was seen between the groups (P = 0.783 and P = 0.648, respectively; Table 5).
      Table 5Reproductive outcome for the four treatment groups
      Parameter5000 IU HCG + 17α- hydroxyprogesterone (n = 21)6500 IU HCG + 17α- hydroxyprogesterone (n = 22)10,000 IU HCG + 17α- hydroxyprogesterone (n = 25)6500 IU HCG + progesterone (n = 26)P-value
      Elevated β-HCG ≥10 IU/l9 (43)8 (36)11 (44)12 (46)0.923
      Positive pregnancy test5 (24)7 (32)8 (32)10 (38)0.783
      Clinical pregnancy4 (19)4 (18)7 (28)7 (27)0.648
      Data are presented as n (%).
      HCG, human chorionic gonadotrophin.

      Serum progesterone adjusted for BMI and age

      Overall, the progesterone concentration adjusted for age showed that for every year of age of the participant the progesterone concentration decreased by 1.96 nmol/l, although this was not statistically significant (P = 0.155).
      A negative significant correlation was found between BMI and progesterone concentration; the latter decreased by 4.05 nmol/l for every 1 kg/m2 increase in BMI (P = 0.005).

      Serum progesterone and 17α-hydroxyprogesterone adjusted for pregnancy

      As expected, pregnant women had a significantly higher progesterone concentration during their last two visits: oocyte retrieval + 10 days (P < 0.001) and oocyte retrieval + 14 days (P < 0.001). The differences in progesterone concentration among pregnant and non-pregnant women are presented in Figure 2b and C. After adjusting the progesterone values for pregnancy, there was still a significant difference in progesterone values (Table 4).

      Serum progesterone and 17α-hydroxyprogesterone adjusted for follicle and oocyte count

      There was a strong positive correlation between follicle count and luteal serum progesterone concentration. Follicles ≥12 mm produced a mean of 9.62 nmol/l progesterone, and each follicle ≥14 mm contributed an additional 1.48 nmol/l progesterone, adjusted for study group and day of visit.
      The strongest association was seen on oocyte retrieval + 4 days, where each follicle ≥12 mm produced 28.1 nmol/l progesterone, and each follicle ≥14 mm produced 29.1 nmol/l. The correlation between follicles ≥12 mm and progesterone concentration was statistically significant on oocyte retrieval + 2 days (P < 0.001), +4 days (P < 0.001), +6 days (P < 0.001), +8 days (P = 0.031) and +14 days (P = 0.035). For follicles ≥14 mm the correlation was significant on oocyte retrieval + 2 days (P < 0.001), +4 days (P < 0.001), +6 days (P < 0.001) and +8 days (P = 0.021).
      Progesterone concentrations between the groups were adjusted separately for follicle count ≥12 mm, ≥14 mm and the two combined. Statistically significant differences in progesterone concentrations were expanded between more groups and on more days when adjusting for follicles ≥12 mm and both ≥12 mm and ≥14 mm (Table 4).
      Exogenous vaginal progesterone administration in group 4 from oocyte retrieval until oocyte retrieval + 14 days resulted in an average increase in serum progesterone of 23.4 nmol/l concentration compared with group 2 (P = 0.212). On oocyte retrieval + 6 days the progesterone difference between group 2 and 4 was 25.8 nmol/l (P = 0.347). Following adjustment for the number of follicles, exogenous progesterone from oocyte retrieval until oocyte retrieval + 14 days in group 4 on average contributed 33.0 nmol/l to the progesterone concentration, showing a significant difference in serum progesterone concentration between group 2 and 4 (P = 0.035).

      Progesterone production related to follicle size at visit oocyte retrieval +2 to +8 days

      In total, from oocyte retrieval + 2 days to oocyte retrieval + 8 days, one corpus luteum ≥12 mm produced on average 17.98 nmol/l progesterone. One follicle ≥14 mm produced an additional 2.36 nmol/l progesterone, giving a total of 20.34 nmol/l progesterone. The progesterone contribution from one additional corpus luteum measuring ≥12 mm (P < 0.001) or ≥14 mm (P < 0.001) was significant.
      The contribution of the corpus luteum to the endogenous progesterone was mainly significant on oocyte retrieval + 2 days to oocyte retrieval + 8 days. From oocyte retrieval + 2 days to oocyte retrieval + 8 days one additional follicle ≥12 mm contributed with 13.57 nmol/l progesterone in group 1, 15.36 nmol/l in group 2, 22.19 nmol/l in group 3, and 19.46 nmol/l in group 4. Furthermore, from oocyte retrieval + 2 days to oocyte retrieval + 8 days one extra follicle ≥14 mm contributed with 10.90 nmol/l progesterone in group 1, 23.49 nmol/l in group 2, 28.58 nmol/l in group 3, and 16.93 nmol/l in group 4. A significant difference in progesterone production from one follicle ≥14 mm was seen between group 1 and 3 (P = 0.007). In group 3 one follicle produced 17.68 nmol/l more progesterone than one follicle in group 1.

      Adverse events

      Serious adverse events and adverse events were recorded throughout the study period, from randomization to pregnancy tests. If there was a positive pregnancy test, the patient was followed until final ultrasonography with confirmation of an intrauterine clinical pregnancy. Where there were side effects, participants were followed until the symptoms disappeared. Only minor local adverse events and, as expected, OHSS were related to the medication. All adverse events were of short duration. All patients with adverse events continued the study, except for one woman who had to discontinue the study due to a potential local allergic reaction after intramuscular progesterone injection.

      Discussion

      To the authors’ knowledge, this is the first study to explore the impact of three different trigger doses of HCG on endogenous progesterone production during the luteal phase of an IVF cycle.
      There was a significant difference in luteal phase serum progesterone concentrations between the three HCG trigger groups (5000 IU, 6500 IU and 10,000 IU HCG). Luteal progesterone concentration increased with increasing HCG trigger dosing, and significant differences were seen during the mid and late luteal phases. Furthermore, the higher the HCG trigger dose, the higher the progesterone output from the individual follicle.
      The results of the present study suggest a positive association between the HCG trigger dose and the luteal progesterone concentration. Importantly, the design of the present study is unique, because, in addition to different HCG trigger doses, the intervention groups received intramuscular 17α-hydroxyprogesterone as LPS to distinguish endogenous progesterone production from exogenous vaginal progesterone support. In all previous studies, participants received a standard LPS that could not be distinguished from endogenous progesterone production (
      • Chan C.C.
      • Ng E.H.
      • Tang O.S.
      • Yeung W.S.
      • Lau E.Y.
      • Ho P.C.
      A prospective, randomized, double-blind study to compare two doses of recombinant human chorionic gonadotropin in inducing final oocyte maturity and the hormonal profile during the luteal phase.
      ;
      • Driscoll G.L.
      • Tyler J.P.
      • Hangan J.T.
      • Fisher P.R.
      • Birdsall M.A.
      • Knight D.C.
      A prospective, randomized, controlled, double-blind, double-dummy comparison of recombinant and urinary HCG for inducing oocyte maturation and follicular luteinization in ovarian stimulation.
      ;
      Group, I.R.H.C.G.S.
      Induction of ovulation in World Health Organization group II anovulatory women undergoing follicular stimulation with recombinant human follicle-stimulating hormone: a comparison of recombinant human chorionic gonadotropin (rhCG) and urinary hCG.
      ;
      • Kovacs P.
      • Kovats T.
      • Bernard A.
      • Zadori J.
      • Szmatona G.
      • Kaali S.G.
      Comparison of serum and follicular fluid hormone levels with recombinant and urinary human chorionic gonadotropin during in vitro fertilization.
      ). As a result, the progesterone concentrations in the present study directly reflect the function of the corpus luteum.
      During the early luteal phase progesterone concentrations were similar between the groups. This was also reported by Chan and colleagues, who compared a 6500 IU with a 13,000 IU HCG trigger dose, measuring the progesterone concentration on the day of oocyte retrieval and 2 days post-retrieval, and by Kovasc and co-workers, who reported similar progesterone concentrations on the oocyte retrieval day after triggering with 6500 IU or 7500 IU HCG (
      • Chan C.C.
      • Ng E.H.
      • Tang O.S.
      • Yeung W.S.
      • Lau E.Y.
      • Ho P.C.
      A prospective, randomized, double-blind study to compare two doses of recombinant human chorionic gonadotropin in inducing final oocyte maturity and the hormonal profile during the luteal phase.
      ;
      • Kovacs P.
      • Kovats T.
      • Bernard A.
      • Zadori J.
      • Szmatona G.
      • Kaali S.G.
      Comparison of serum and follicular fluid hormone levels with recombinant and urinary human chorionic gonadotropin during in vitro fertilization.
      ).
      Previously, studies with one or few blood samples taken during the mid-luteal phase reported the same association between HCG dose and progesterone concentration as the present study. Thus, in a long GnRH agonist down-regulation protocol, one study found significantly higher progesterone concentrations at HCG trigger + 10 days with the higher, 13,000 IU HCG, trigger dose compared with 6500 IU HCG (
      • Chan C.C.
      • Ng E.H.
      • Tang O.S.
      • Yeung W.S.
      • Lau E.Y.
      • Ho P.C.
      A prospective, randomized, double-blind study to compare two doses of recombinant human chorionic gonadotropin in inducing final oocyte maturity and the hormonal profile during the luteal phase.
      ). Moreover, Driscoll and associates reported a significantly higher progesterone concentration 6–7 days after the HCG trigger with a 6500 IU compared with a 5000 IU HCG trigger dose (
      • Driscoll G.L.
      • Tyler J.P.
      • Hangan J.T.
      • Fisher P.R.
      • Birdsall M.A.
      • Knight D.C.
      A prospective, randomized, controlled, double-blind, double-dummy comparison of recombinant and urinary HCG for inducing oocyte maturation and follicular luteinization in ovarian stimulation.
      ). Finally, a timed intercourse/insemination study reported a higher progesterone concentration after 6500 IU HCG compared with 5000 IU HCG, 5–7 days after the trigger (
      Group, I.R.H.C.G.S.
      Induction of ovulation in World Health Organization group II anovulatory women undergoing follicular stimulation with recombinant human follicle-stimulating hormone: a comparison of recombinant human chorionic gonadotropin (rhCG) and urinary hCG.
      ).
      In the present study, no difference was seen between trigger groups as regards the number of mature, fertilized oocytes and good-quality embryos. Previous studies have also investigated the relationship between different HCG trigger doses and oocytes, maturity and embryo quality. Thus, a study by Abdalla and co-workers found that 2000 IU HCG used as trigger dose resulted in a significant decrease in the number of oocytes retrieved compared with a 5000 IU and a 10,000 IU HCG trigger dose (
      • Abdalla H.I.
      • Ah-Moye M.
      • Brinsden P.
      • Howe D.L.
      • Okonofua F.
      • Craft I.
      The effect of the dose of human chorionic gonadotropin and the type of gonadotropin stimulation on oocyte recovery rates in an in vitro fertilization program.
      ). Moreover, a more recent study reported that triggering with 4000 IU HCG, compared with 6000 IU HCG, did not affect the number of mature oocytes, although it resulted in a lower fertilization rate and a lower pregnancy rate (
      • Lin H.
      • Wang W.
      • Li Y.
      • Chen X.
      • Yang D.
      • Zhang Q.
      Triggering final oocyte maturation with reduced doses of hCG in IVF/ICSI: a prospective, randomized and controlled study.
      ). In contrast, a retrospective cohort study in high-responder patients receiving 3300 IU or 5000 IU for triggering found similar numbers of mature and fertilized oocytes, and no difference in pregnancy rates (
      • Schmidt D.W.
      • Maier D.B.
      • Nulsen J.C.
      • Benadiva C.A.
      Reducing the dose of human chorionic gonadotropin in high responders does not affect the outcomes of in vitro fertilization.
      ). Finally, a retrospective cohort study using a 3300 IU, 4000 IU, 5000 IU or 10,000 IU HCG trigger dose reported no significant differences in oocyte maturity, clinical pregnancy or live birth rate between the groups (
      • Gunnala V.
      • Melnick A.
      • Irani M.
      • Reichman D.
      • Schattman G.
      • Davis O.
      • Rosenwaks Z.
      Sliding scale HCG trigger yields equivalent pregnancy outcomes and reduces ovarian hyperstimulation syndrome: Analysis of 10,427 IVF-ICSI cycles.
      ).
      The findings of the present and previous studies suggest that an HCG trigger dose of 5000 IU secures the retrieval of mature oocytes and fertilization. However, from the current data, it appears that, to obtain the optimal peri-implantation progesterone concentration in a woman with no risk of OHSS, a trigger dose higher than 5000 IU HCG should be used if a fresh transfer is planned.
      Implantation occurs around 7 days after oocyte retrieval, and therefore the progesterone profile around that period is of particular importance (
      • Navot D.
      • Scott R.T.
      • Droesch K.
      • Veeck L.L.
      • Liu H.C.
      • Rosenwaks Z.
      The window of embryo transfer and the efficiency of human conception in vitro.
      ). As early as 1982 Hull and colleagues reported that, in a natural cycle, there is an optimal progesterone range for pregnancy during the luteal phase with lower and upper limits of 27 nmol/l and 53 nmol/l (
      • Hull M.G.
      • Savage P.E.
      • Bromham D.R.
      • Ismail A.A.
      • Morris A.F.
      The value of a single serum progesterone measurement in the midluteal phase as a criterion of a potentially fertile cycle ("ovulation") derived form treated and untreated conception cycles.
      ). Moreover, a recent large cohort study showed that, to obtain the highest chance of live birth in fresh transfer IVF cycles, the optimal progesterone concentration (2 or 3 days after oocyte retrieval) is 60–100 nmol/l during the early luteal phase and 150–250 nmol/l during the mid-luteal phase (oocyte retrieval + 5 days) (
      • Thomsen L.H.
      • Kesmodel U.S.
      • Erb K.
      • Bungum L.
      • Pedersen D.
      • Hauge B.
      • Elbæk H.O.
      • Povlsen B.B.
      • Andersen C.Y.
      • Humaidan P.
      The impact of luteal serum progesterone levels on live birth rates-a prospective study of 602 IVF/ICSI cycles.
      ).
      During the early luteal phase of the present study (oocyte retrieval + 2 days and + 4 days) the progesterone concentrations were within the optimal range in all the groups. In contrast, 6 days after oocyte retrieval, group 1, triggered with 5000 IU HCG, had a median progesterone concentration of 61.7 nmol/l, significantly below the suggested 150–250 nmol/l optimal progesterone concentration (
      • Thomsen L.H.
      • Kesmodel U.S.
      • Andersen C.Y.
      • Humaidan P.
      Daytime Variation in Serum Progesterone During the Mid-Luteal Phase in Women Undergoing In Vitro Fertilization Treatment.
      ). When compared separately with the three other study groups, progesterone concentrations in group 1 were significantly lower. Although this study was not powered for reproductive outcomes, a lower clinical pregnancy rate was seen in group 1 and 2, in which the progesterone concentration first decreased considerably from oocyte retrieval + 6 days.
      One of the major functions of the corpus luteum is the production of progesterone, which secures the receptivity of the endometrium and supports the early implant (
      • Devoto L.
      • Fuentes A.
      • Kohen P.
      • Céspedes P.
      • Palomino A.
      • Pommer R.
      • Muñoz A.
      • Strauss 3rd, J.F.
      The human corpus luteum: life cycle and function in natural cycles.
      ). In a natural cycle the progesterone concentration peaks above 30 nmol/l from 5–10 days after ovulation before it decreases (
      • Groome N.P.
      • Illingworth P.J.
      • O'Brien M.
      • Pai R.
      • Rodger F.E.
      • Mather J.P.
      • McNeilly A.S.
      Measurement of dimeric inhibin B throughout the human menstrual cycle.
      ). In a recent study, a median peak progesterone concentration of 114 ng/ml was reported at oocyte retrieval + 4 days in IVF patients triggered with 6500 IU HCG in a freeze-all protocol without luteal support (
      • Vuong L.N.
      • Ho T.M.
      • Pham T.D.
      • Ho V.N.A.
      • Andersen C.Y.
      • Humaidan P.
      The early luteal hormonal profile in IVF patients triggered with hCG.
      ). The present study showed a comparable peak 4 days after oocyte retrieval in all the groups. The peak concentration of progesterone 4 days after oocyte retrieval was 228 nmol/l in the group that was triggered with 6500 IU HCG and received 17α-hydroxyprogesterone for LPS (group 2). Moreover, the progesterone concentration decreased first rapidly from oocyte retrieval + 4 days to + 6 days in group 1 and shortly thereafter in group 2, while the progesterone concentration decreased later in group 3 and 4 (10,000 IU HCG and 6500 IU HCG).
      The explanation for the slower decline in progesterone concentration in group 3 compared with group 1 and 2 is obviously the higher HCG dose. This finding is consistent with earlier observations showing that a 5000 IU HCG trigger dose is ‘cleared’ after 7 days, whereas a 10,000 IU HCG trigger dose is detectable in serum 9–10 days after administration (
      • Weissman A.
      • Lurie S.
      • Zalel Y.
      • Goldchmit R.
      • Shoham Z.
      Human chorionic gonadotropin: pharmacokinetics of subcutaneous administration.
      ). Those early findings have subsequently been corroborated by others (
      • Trinchard-Lugan I.
      • Khan A.
      • Porchet H.C.
      • Munafo A.
      Pharmacokinetics and pharmacodynamics of recombinant human chorionic gonadotrophin in healthy male and female volunteers.
      ). In conclusion, a 10,000 IU HCG trigger dose induces a longer and more sustained support of the corpus luteum and thus nearer to an optimal progesterone concentration around implantation.
      A novel finding of the present study is the fact that the progesterone production from every single follicle increased significantly as the HCG trigger dose was increased. Importantly, it shows that the progesterone production potential of the corpus luteum is not fully utilized when a 5000 IU trigger dose is used. One explanation could be that not all LH receptors on the corpus luteum are occupied using the lower HCG dose, resulting in sub-optimal production. However, the finding is in line with natural conception in which increasing HCG production by the trophoblast during early pregnancy boosts the corpus luteum to increase its progesterone production (
      • Devoto L.
      • Fuentes A.
      • Kohen P.
      • Céspedes P.
      • Palomino A.
      • Pommer R.
      • Muñoz A.
      • Strauss 3rd, J.F.
      The human corpus luteum: life cycle and function in natural cycles.
      ;
      • Yovich J.L.
      • Stanger J.D.
      • Yovich J.M.
      • Tuvik A.I.
      • Turner S.R.
      Hormonal profiles in the follicular phase, luteal phase and first trimester of pregnancies arising from in-vitro fertilization.
      ). An unanswered question is whether a 10,000 IU HCG trigger dose reflects the true potential of the corpus luteum or whether a higher HCG trigger dose would further increase the progesterone output.
      The most direct measure of corpus luteum function is the endogenous progesterone concentration. In reviewing the literature, the current authors were not able to find any description of the association between the HCG trigger dose and the corpus luteum function estimated by the endogenous progesterone concentration from follicles of ≥12 mm. As mentioned, all follicles that were ≥12 mm on the trigger day were measured. The assumption was that follicles ≥12 mm in size have the capacity to convert into a competent corpus luteum and will contribute to endogenous progesterone production. Owing to the unique design of the study, it was possible to estimate that one follicle ≥12 mm resulted in on average 9.6 nmol/l progesterone during the whole luteal phase. At oocyte retrieval + 4 days, the endogenous progesterone production from one corpus luteum was highest, with an average of 28.1 nmol/l progesterone. Due to the fact that one follicle could significantly influence the progesterone concentration, the differences in progesterone concentration between the groups were adjusted for follicle number. However, even after adjusting for follicle number the significant differences in the progesterone concentration among the groups remained constant. Finally, another interesting finding was that from 2 to 8 days after oocyte retrieval, each follicle ≥14 mm in size produced 17.7 nmol/l more progesterone in group 3 (10,000 IU) compared with group 1 (5000 IU; P = 0.007). This new finding is clinically relevant, suggesting that increasing the HCG trigger dose in women with no risk of OHSS will boost each corpus luteum to produce more progesterone.
      The strengths and limitations of this study also need to be discussed. The major strength of this study is the randomized controlled trial design with well-defined inclusion and exclusion criteria, complete follow-up, close monitoring using eight blood samples during the luteal phase, and an almost complete dataset.
      A limitation of the study is the moderate number of patients included; however, this was partly compensated for by the large number of blood samples obtained from each participant. Admittedly, the progesterone concentration could be influenced by circadian fluctuations, but a recent luteal-phase study showed that low progesterone concentrations (<60 nmol/l) remain low and high concentrations remain high during the daytime (
      • Thomsen L.H.
      • Kesmodel U.S.
      • Andersen C.Y.
      • Humaidan P.
      Daytime Variation in Serum Progesterone During the Mid-Luteal Phase in Women Undergoing In Vitro Fertilization Treatment.
      ). An additional uncontrolled factor was that uHCG production was discontinued by the manufacturer. Consequently, all doses were changed to comparable rHCG doses, as the pharmacodynamics of rHCG and uHCG are similar (
      • Trinchard-Lugan I.
      • Khan A.
      • Porchet H.C.
      • Munafo A.
      Pharmacokinetics and pharmacodynamics of recombinant human chorionic gonadotrophin in healthy male and female volunteers.
      ). The small number of cases does not allow a thorough subgroup analysis. Moreover, the study was unblinded. Finally, the authors acknowledge that ultrasound follicle measurement in IVF can be prone to variations between observers, but the ultrasonography was limited to only a few experienced doctors.
      In conclusion, this study shows that a higher HCG trigger dose induces a higher progesterone concentration during the mid–late luteal phase. Moreover, a significant relationship was found between the trigger dose and the progesterone output from each individual follicle. Although more research is needed, this new knowledge suggests that the HCG trigger dose should be individualized in IVF to secure an optimal peri-implantation progesterone concentration and subsequently an optimal live birth rate.

      Acknowledgements

      The authors thank all the infertile couples who participated in this study. Moreover, they thank the doctors, nurses and laboratory staff from the Fertility Clinic, Odense University Hospital for their active participation in the study. The authors acknowledge OPEN and the biochemical department at Odense University Hospital for assistance in biobanking and analysing blood samples. Finally, thanks goes to IBSA and NordicInfu Care for providing Lentogest.

      Funding

      An unrestricted grant was given by NordicInfu Care, Sweden, and grants were received from the University of South of Denmark, Region of South of Denmark, OPEN, and the Research Fund of chief doctor committee OUH. The funding providers were not involved in conducting the study or writing or approval of the manuscript.

      Appendix. Supplementary materials

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      Biography

      Louise Svenstrup obtained her MD degree in 2008. She currently works as a PhD student and sometimes at the Fertility Clinic, Odense University Hospital, Denmark with Peter Humaidan and Jens Fedder as promoters. Her research interests include ovulation and reproductive endocrinology, especially progesterone and HCG.
      Key message
      A dose–response relationship exists between the HCG trigger dose and total endogenous progesterone production during the mid–late luteal phase. Moreover, a higher HCG trigger dose increases the progesterone output from the individual follicle. This suggests the HCG trigger dose should be individualized in IVF to secure the optimal peri-implantation progesterone concentration.