Advertisement

Outcome of vitrified-warmed blastocyst transfer performed on days 5–7 after urine LH detection

  • Vida Gavrić Lovrec
    Affiliations
    Department of Reproductive Medicine and Gynaecological Endocrinology, Division of Gynaecology and Perinatology, University Medical Centre Maribor, Ljubljanska Maribor, Slovenia
    Search for articles by this author
  • Nejc Kozar
    Correspondence
    Corresponding author.
    Affiliations
    Department of Reproductive Medicine and Gynaecological Endocrinology, Division of Gynaecology and Perinatology, University Medical Centre Maribor, Ljubljanska Maribor, Slovenia
    Search for articles by this author
  • Milan Reljic
    Affiliations
    Department of Reproductive Medicine and Gynaecological Endocrinology, Division of Gynaecology and Perinatology, University Medical Centre Maribor, Ljubljanska Maribor, Slovenia
    Search for articles by this author
Open AccessPublished:December 12, 2021DOI:https://doi.org/10.1016/j.rbmo.2021.12.008

      Abstract

      Research question

      What is the clinical importance of vitrified-warmed blastocyst transfer timing if performed on days 5, 6 and 7 after detecting the LH surge using urine tests?

      Design

      Between 2013 and 2019, 2080 vitrified-warmed blastocyst transfers in a true natural cycle were performed and later analysed at the Department of Reproductive Medicine, University Medical Centre Maribor, Slovenia. Urine LH tests were performed twice daily to monitor the onset of the LH surge. Vitrified-warmed blastocyst transfer (frozen embryo transfer [FET]) was performed on day 5 (group 1), 6 (group 2) or 7 (group 3) after the LH surge in 18.3%, 77.4% and 4.3% of cycles, respectively. The patient and cycle characteristics among the groups were compared using the Cochran–Mantel–Haenszel test and respective generalized linear mixed models. Propensity score matching was used to adjust for potential differences among the groups.

      Results

      There were no statistically significant differences between groups 1, 2 and 3 in the cycle and patient characteristics, clinical pregnancy rate (37.6% versus 39.3% versus 31.1%), implantation rate (34.3% versus 35.6% versus 30.9%), miscarriage rate (7.1% versus 8.6% versus 6.7%) and delivery rate (30.5% versus 30.7% versus 24.4%). The day of FET after the LH surge detected using a urine test was not significantly associated with live births.

      Conclusions

      The results of the current study suggested that the vitrified-warmed blastocyst transfer could be scheduled on day 5, 6 or 7 after a positive LH urine test without having a significant impact on the clinical outcome.

      Key words

      Introduction

      In recent years, vitrified-warmed blastocyst transfer (frozen embryo transfer [FET]) has become an important part of IVF programmes. Elective single-embryo transfer and the freeze-all strategy, together with advanced cryopreservation techniques, provide important benefits for women and primarily improve the safety of the treatment and produce a higher cumulative live birth rate. Various endometrial preparation strategies have been introduced to optimize the success of FET; however, the optimal method has not yet been established. The most frequently used cycle regimens include a natural ovulatory cycle protocol with LH detection (true natural cycle [tNC]), human chorionic gonadotrophin (HCG) application (modified natural cycle [mNC]) and artificial cycles. Each has advantages and disadvantages. The benefit of natural cycle FET is the avoidance of medication, and the disadvantage is the need for precise monitoring and limited flexibility during the timing of FET (
      • Reljič M.
      • Knez J.
      Predicted luteal phase length has no influence on success of vitrified-warmed blastocyst transfer in natural cycle.
      ).
      In tNC, the timing of FET is determined after the spontaneous LH surge and/or detection of ovulation. FET should be performed when the endometrium is receptive during the window of implantation (WOI). In the textbook 28-day cycle, the WOI opens on day 19 and remains open for 4–5 days in the endometrial cycle at the time of maximal serum progesterone concentration (
      • Lessey B.A.
      • Young S.L.
      What exactly is endometrial receptivity?.
      ;
      • Navot D.
      • Bergh P.A.
      • Williams M.
      • Garrisi G.J.
      • Guzman I.
      • Sandler B.
      • Fox J.
      • Schreiner-Engel P.
      • Hofmann G.E.
      • Grunfeld L.
      An insight into early reproductive processes through the in vivo model of ovum donation.
      ;
      • Psychoyos A.
      Uterine receptivity for nidation.
      ). Vitrified-warmed blastocyst transfer on day 6 after the LH surge has been proposed to achieve a synchronization between embryonic and endometrial development and optimal implantation rate (
      • Mackens S.
      • Santos-Ribeiro S.
      • van de Vijver A.
      • Racca A.
      • Van Landuyt L.
      • Tournaye H.
      • Blockeel C.
      Frozen embryo transfer: a review on the optimal endometrial preparation and timing.
      ).
      The LH surge usually begins between midnight and 07:30 hours in two-thirds of women; consequently, follicle rupture occurs 34–36 h later, while the LH surge itself lasts 48–50 h (
      • Glass R.H.
      • Speroff L.
      Clinical gynecologic endocrinology and infertility.
      ;
      • Hoff J.D.
      • Quigley M.E.
      • Yen S.S.
      Hormonal dynamics at midcycle: a reevaluation.
      ). As urine LH increases with a delay of 12–36 h after the detection of the blood LH surge, synchronization could also be achieved if FET were performed on day 5 after a positive LH urine test (
      • Martinez F.
      • Trounson A.
      • Besanko M.
      Detection of the LH surge for AID, AIH and embryo transfer using a twice daily urinary dip-stick assay.
      ). However, there are currently no studies in the literature on this topic.
      This study aimed to establish whether the clinical outcomes of vitrified-warmed blastocyst transfer were comparable if FET was performed on day 5, 6 or 7 after LH surge detection using urine tests.

      Material and methods

      All vitrified-warmed blastocyst transfers performed in a tNC between 2013 and 2019 at the Department of Reproductive Medicine, University Medical Centre Maribor, were included in this retrospective study. Women with uterine pathology, a hydrosalpinx visible on ultrasound or an endocrinological disorder, such as polycystic ovarian syndrome or premature ovarian insufficiency, were excluded from the analysis.
      Women below 43 years of age with regular menstrual cycles (24–35 days) were included in the study. Ovulatory status was confirmed during a diagnostic workup (the dominant follicle in the preovulatory phase on ultrasound and progesterone in the mid-luteal phase).

       Cycle monitoring

      Vaginal ultrasound was performed in all participants on day 8–10 of the cycle. If the leading follicle was selected and accompanied by an adequate thickening and ultrasonographic appearance of the endometrium, the patient was instructed to perform a urine LH test (RapiTest LH; MD Doctors Direct, Switzerland) every morning from the day of the anticipated follicular diameter of 15 mm. An increase of the follicle diameter by 2 mm/day was assumed. When a woman had a positive LH test in the morning, she was instructed to repeat the test in the evening of the same day to confirm the LH surge. If the LH surge was undetected or if the results were inconclusive, the FET was cancelled. FET were routinely performed on day 6 after the LH surge and could be carried out on any day of the week. Occasionally, FET was scheduled on day 5 or 7 after the LH surge to avoid transfer on busy days or to modify the workload for the staff at weekends.

       Blastocyst warming and evaluation

      All expanded blastocysts were vitrified on days 5 or 6 using a combination of dimethyl sulfoxide and ethylene glycol cryoprotectants. Before vitrification, blastocysts were graded according to the centre's established grading system (
      • Kovačič B, V.V.
      Importance of blastocyst morphology in selection for transfer.
      ;
      • Kovacic B.
      • Vlaisavljevic V.
      • Reljic M.
      • Cizek-Sajko M.
      Developmental capacity of different morphological types of day 5 human morulae and blastocysts.
      ;
      • Martinez F.
      • Trounson A.
      • Besanko M.
      Detection of the LH surge for AID, AIH and embryo transfer using a twice daily urinary dip-stick assay.
      ). After thawing, blastocysts were cultured in a recovery medium (Blast Assist System; Origio, Denmark) for at least 4 h before transfer to the uterus. Only blastocysts that had at least 50% intact blastomeres after thawing and the start of re-expansion were assessed as suitable for transfer (
      • Kovačič B, V.V.
      Importance of blastocyst morphology in selection for transfer.
      ). Preimplantation genetic testing for aneuploidy was not performed in the included cycles.
      One or two vitrified-warmed blastocysts were transferred using Labotect catheters (Labotect Labor-Technik-Göttingen, Germany). The number of embryos transferred in each case depended on the quality of the available embryos, the number of previous treatments, the number of embryos vitrified in the same straw and the patient–doctor agreement.

       Embryo transfer and outcome

      A pelvic ultrasound examination was performed immediately before embryo transfer to measure the endometrial thickness and evaluate the endometrial pattern.
      Progesterone supplementation (400 mg/day of micronized vaginal progesterone) was initiated immediately after FET. The serum HCG test was performed 2 weeks after FET, and clinical pregnancy, defined as ultrasonographic documentation of at least one fetus with a discernible heartbeat, was evaluated 2 weeks later. The clinical pregnancy rate was calculated as the number of clinical pregnancies divided by the number of transfer cycles. Similarly, the implantation rate was calculated as the number of gestational sacs observed divided by the number of embryos transferred. Both values were expressed as percentages. Miscarriage was defined as the number of spontaneous losses of clinical pregnancies before 22 completed weeks of gestation divided by the total number of clinical pregnancies. Additionally, the live birth rate was calculated as the number of deliveries that resulted in at least one live birth per all embryo transfers.
      Patient characteristics and clinical data were collected from the authors’ software database.

       Statistical analyses

      Reproductive outcomes and cycle and patient characteristics of FET on days 5, 6 and 7 after the LH surge were compared using the Cochran–Mantel–Haenszel test for factorial variables. Power and sample size calculations for the Cochran–Mantel–Haenszel test were performed. According to literature-based success rates, 58, 253 and 16 cycles were required on days 5, 6 and 7, respectively, for the desired power of 0.9. Univariate analysis was performed to consider the effect of successive cycles by building respective generalized linear models with the couples’ unique ID as a blocking variable. Furthermore, the three groups were propensity score matched to minimize potential bias (Figure 1). The propensity scores were calculated for all pairs of the three groups using binary logistic regression based on the woman's age, number of transferred embryos and blastocyst quality.
      Figure 1
      Figure 1The distribution of propensity scores before and after propensity score matching.
      Success rates were compared using the Cochran–Mantel–Haenszel test. Statistical analyses were performed with RStudio using R version 4.0.3 (R Core Team 2020; R: Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Austria).

       Ethical approval and consent to participate

      As all the data were collected as part of quality control with no direct influence on the treatment, ethical approval was not required.

      Results

      A total of 2731 FET were performed. After removing missing data, 2080 vitrified-warmed blastocyst transfers, performed in 1707 couples, were included in the analysis. Following positive LH urine tests, 1610 (77.4%), 380 (18.3%) and 90 (4.3%) FET were performed on days 5, 6 and 7. Differences in women's age, cause of infertility, number of previous IVF attempts, medical history regarding previous IVF cycles and their outcome, average menstrual cycle length and menstrual cycle variability were not observed. Furthermore, no differences were found in the FET cycle characteristics on the day of the LH surge, endometrial thickness on the day of FET and endometrial morphology (Table 1). The number of blastocysts transferred in all groups was the same; the proportion of difficult embryo transfers, the number of transferred blastocysts vitrified on day 5 and the proportion of morphologically optimal blastocyst transfers also did not differ among the cycles with FET on days 5 and 6 and 7 after the LH surge (Table 1). Women undergoing FET on day 5, 6 or 7 after the LH surge showed no statistically significant differences concerning clinical pregnancy rate, implantation rate, miscarriage rate or live birth rate (Table 2). Propensity score matching was performed using age, number of transferred embryos and blastocyst quality to account for potential differences among the groups; the Cochran–Mantel–Haenszel test was used to compare FET cycle outcomes between the respective groups. No statistical difference was found in live birth rates (day 5 versus day 6, P = 0.98; day 6 versus day 7, P = 0.31; day 5 versus day 7 P = 0.40).
      Table 1Comparison of patient and cycle characteristics between vitrified-warmed blastocyst transfers performed on days 5, 6 and 7 after the urine LH surge
      ParameterFET after the LH surge
      Day 5Day 6Day 7
      Number of transfers380161090
      Age (years), median (IQR)34 (31–37)34 (27 –41)34 (27–41)
      Unexplained infertility, n (%)168 (44)658 (41)32 (36)
      Tubal factor infertility, n (%)66 (17)310 (19)20 (22)
      Male factor infertility, n (%)153 (40)669 (42)35 (39)
      No. of previous IVF/ICSI cycles, median (IQR)2 (1–2)2 (1–3)2 (1–3)
      Proportion of cycles with birth after a fresh embryo transfer, n (%)65 (17)279 (17)8 (9)
      Proportion of FET after freeze-all cycles, n (%)48 (13)200 (12)17 (19)
      Menstrual cycle length (days), median (IQR)28 (28–29)28 (27–29)28 (27–29)
      Menstrual cycle variability (days), median (IQR)3 (2–5)4 (1–7)5 (1–9)
      Day of the LH surge using a urine LH test, median (IQR)13 (12–15)13 (10–16)13 (9–17)
      Endometrial thickness on the day of FET (mm), median (IQR)10 (8–11)10 (7–13)10 (8–12)
      Secretory endometrium pattern, n (%)203 (53)978 (61)57 (63)
      No. of blastocysts transferred, median (IQR)1 (1–2)1 (1–2)1 (1–2)
      Proportion of difficult embryo transfers, n (%)27 (7)80 (5)3 (3)
      Proportion of blastocysts vitrified on day 5, n (%)226 (59)982 (61)62 (69)
      Proportion of embryo transfers of morphologically optimal blastocysts, n (%)66 (17)258 (16)12 (13)
      FET, vitrified-warmed blastocyst transfer; ICSI, intra-cytoplasmic sperm injection; IQR, interquartile range.
      Table 2Comparison of outcomes between vitrified-warmed blastocyst transfers performed on days 5, 6 and 7 after the urine LH surge
      ParameterFET after the LH surge
      Day 5Day 6Day 7
      N380161090
      Clinical pregnancy rate, n (%)143 (38)633 (39)28 (31)
      Implantation rate, implanted/transferred, n/N (%)156/456 (34)681/1966 (35)30/97 (31)
      Miscarriage rate, n (%)27 (7)138 (9)6 (7)
      Live birth rate, n (%)116 (31)495 (31)22 (24)
      FET, vitrified-warmed blastocyst transfer.

      Discussion

      The number of FET cycles performed in recent years has drastically increased due to the trend of transferring fewer embryos in a fresh IVF cycle and to improved laboratory techniques (
      • Groenewoud E.R.
      • Cantineau A.E.P.
      • Kollen B.J.
      • Macklon N.S.
      • Cohlen B.J.
      What is the optimal means of preparing the endometrium in frozen-thawed embryo transfer cycles? A systematic review and meta-analysis.
      ). In a spontaneous cycle in women with regular menstrual cycles, FET seems to be a reasonable choice because of good results and a lack of medication requirement before FET. Vaginal progesterone supplementation is suggested after FET in tNC (
      • Casper R.F.
      • Yanushpolsky E.H.
      Optimal endometrial preparation for frozen embryo transfer cycles: window of implantation and progesterone support.
      ). tNC FET is the preferred approach to FET in women with regular menstrual cycles at the authors’ centre, and 2731 FETs in tNC were performed between 2013 and 2019. The problem of FETs in a natural cycle is the lack of flexibility.
      FET timing in natural cycles can be determined by the detection of spontaneous LH or ovulation induction using HCG. In the latter case (mNC), careful cycle monitoring using ultrasonography and hormone assessment to identify the endometrial receptive period is required. HCG is usually administered when the dominant follicle reaches a size indicating its maturity (16–20 mm), considering or regardless of LH and progesterone concentrations (
      • Casper R.F.
      • Yanushpolsky E.H.
      Optimal endometrial preparation for frozen embryo transfer cycles: window of implantation and progesterone support.
      ;
      • Greco E.
      • Litwicka K.
      • Arrivi C.
      • Varricchio M.T.
      • Caragia A.
      • Greco A.
      • Minasi M.G.
      • Fiorentino F.
      The endometrial preparation for frozen-thawed euploid blastocyst transfer: a prospective randomized trial comparing clinical results from natural modified cycle and exogenous hormone stimulation with GnRH agonist.
      ;
      • Groenewoud E.R.
      • Cantineau A.E.P.
      • Kollen B.J.
      • Macklon N.S.
      • Cohlen B.J.
      What is the optimal means of preparing the endometrium in frozen-thawed embryo transfer cycles? A systematic review and meta-analysis.
      ,
      • Groenewoud E.R.
      • Kollen B.J.
      • Macklon N.S.
      • Cohlen B.J.
      Spontaneous LH surges prior to HCG administration in unstimulated-cycle frozen-thawed embryo transfer do not influence pregnancy rates.
      ). Earlier data speak against the use of HCG because of the initially unsatisfying results; however, recent studies have not confirmed earlier conclusions, although there might be a benefit of tNC attributed to LH-induced changes in the endometrium that favour implantation (
      • Fatemi H.M.
      • Kyrou D.
      • Bourgain C.
      • Van den Abbeel E.
      • Griesinger G.
      • Devroey P.
      Cryopreserved-thawed human embryo transfer: spontaneous natural cycle is superior to human chorionic gonadotropin-induced natural cycle.
      ;
      • Groenewoud E.R.
      • Cohlen B.J.
      • Al-Oraiby A.
      • Brinkhuis E.A.
      • Broekmans F.J.M.
      • de Bruin J.P.
      • van den Dool G.
      • Fleisher K.
      • Friederich J.
      • Goddijn M.
      • Hoek A.
      • Hoozemans D.A.
      • Kaaijk E.M.
      • Koks C.A.M.
      • Laven J.S.E.
      • van der Linden P.J.Q.
      • Manger A.P.
      • Slappendel E.
      • Spinder T.
      • Kollen B.J.
      • Macklon N.S.
      A randomized controlled, non-inferiority trial of modified natural versus artificial cycle for cryo-thawed embryo transfer.
      ;
      • Huberlant S.
      • Vaast M.
      • Anahory T.
      • Tailland M.L.
      • Rougier N.
      • Ranisavljevic N.
      • Hamamah S.
      Natural cycle for frozen-thawed embryo transfer: Spontaneous ovulation or triggering by HCG.
      ;
      • Kyrou D.
      • Kolibianakis E.M.
      • Fatemi H.M.
      • Grimbizis G.F.
      • Theodoridis T.D.
      • Camus M.
      • Tournaye H.
      • Tarlatzis B.C.
      • Devroey P.
      Spontaneous triggering of ovulation versus HCG administration in patients undergoing IUI: a prospective randomized study.
      ,
      • Mackens S.
      • Stubbe A.
      • Santos-Ribeiro S.
      • et al.
      To trigger or not to trigger ovulation in a natural cycle for frozen embryo transfer: a randomized controlled trial.
      ). In a tNC, the LH surge is accompanied by an FHS surge, the role of which is still not fully understood; it might influence the function of the corpus luteum through the induction of LH receptors in the luteinizing granulosa cells (
      • Humaidan P.
      • Kol S.
      • Papanikolaou E.G.
      GnRH agonist for triggering of final oocyte maturation: time for a change of practice?.
      ). An early progesterone rise before ovulation probably contributes to the induction of the WOI in a natural cycle (
      • Mackens S.
      • Santos-Ribeiro S.
      • van de Vijver A.
      • Racca A.
      • Van Landuyt L.
      • Tournaye H.
      • Blockeel C.
      Frozen embryo transfer: a review on the optimal endometrial preparation and timing.
      ).
      There is no unanimous definition of an LH surge. Some clinicians define the LH surge as a 180% increase above the basal concentration (
      • Fatemi H.M.
      • Kyrou D.
      • Bourgain C.
      • Van den Abbeel E.
      • Griesinger G.
      • Devroey P.
      Cryopreserved-thawed human embryo transfer: spontaneous natural cycle is superior to human chorionic gonadotropin-induced natural cycle.
      ;
      • Groenewoud E.R.
      • Cohlen B.J.
      • Al-Oraiby A.
      • Brinkhuis E.A.
      • Broekmans F.J.M.
      • de Bruin J.P.
      • van den Dool G.
      • Fleisher K.
      • Friederich J.
      • Goddijn M.
      • Hoek A.
      • Hoozemans D.A.
      • Kaaijk E.M.
      • Koks C.A.M.
      • Laven J.S.E.
      • van der Linden P.J.Q.
      • Manger A.P.
      • Slappendel E.
      • Spinder T.
      • Kollen B.J.
      • Macklon N.S.
      A randomized controlled, non-inferiority trial of modified natural versus artificial cycle for cryo-thawed embryo transfer.
      ;
      • Huberlant S.
      • Vaast M.
      • Anahory T.
      • Tailland M.L.
      • Rougier N.
      • Ranisavljevic N.
      • Hamamah S.
      Natural cycle for frozen-thawed embryo transfer: Spontaneous ovulation or triggering by HCG.
      ;
      • Humaidan P.
      • Kol S.
      • Papanikolaou E.G.
      GnRH agonist for triggering of final oocyte maturation: time for a change of practice?.
      ;
      • Kyrou D.
      • Kolibianakis E.M.
      • Fatemi H.M.
      • Grimbizis G.F.
      • Theodoridis T.D.
      • Camus M.
      • Tournaye H.
      • Tarlatzis B.C.
      • Devroey P.
      Spontaneous triggering of ovulation versus HCG administration in patients undergoing IUI: a prospective randomized study.
      ;
      • Park S.J.
      • Goldsmith L.T.
      • Skurnick J.H.
      • Wojtczuk A.
      • Weiss G.
      Characteristics of the urinary luteinizing hormone surge in young ovulatory women.
      ), whereas others use a serum LH concentration of 10 IU/l or more (
      • Groenewoud E.R.
      • Cantineau A.E.P.
      • Kollen B.J.
      • Macklon N.S.
      • Cohlen B.J.
      What is the optimal means of preparing the endometrium in frozen-thawed embryo transfer cycles? A systematic review and meta-analysis.
      ). An abrupt increase in LH with a doubling time of 2 h, a duration of 48 h, and a second rapid rise of progesterone 36 h after the beginning of the LH surge and 12 h before its termination have been reported (
      • Hoff J.D.
      • Quigley M.E.
      • Yen S.S.
      Hormonal dynamics at midcycle: a reevaluation.
      ). Studies show high variability in the configuration, amplitude and duration of the LH surge (
      • Direito A.
      • Bailly S.
      • Mariani A.
      • Ecochard R.
      Relationships between the luteinizing hormone surge and other characteristics of the menstrual cycle in normally ovulating women.
      ;
      • Park S.J.
      • Goldsmith L.T.
      • Skurnick J.H.
      • Wojtczuk A.
      • Weiss G.
      Characteristics of the urinary luteinizing hormone surge in young ovulatory women.
      ). The LH surge onset precedes ovulation by 34–36 h but sometimes by up to 44 h. For that reason, the timing of FET can be assumed to be flexible (
      • Glass R.H.
      • Speroff L.
      Clinical gynecologic endocrinology and infertility.
      ;
      • Su H.W.
      • Yi Y.C.
      • Wei Ty Chang T.C.
      • Cheng C.M.
      Detection of ovulation,a review of currently available methods.
      ). Makens and colleagues suggested that FET should be performed on days HCG +7 in an mNC and LH +6 in a tNC based on the different time spans to ovulation (
      • Mackens S.
      • Santos-Ribeiro S.
      • van de Vijver A.
      • Racca A.
      • Van Landuyt L.
      • Tournaye H.
      • Blockeel C.
      Frozen embryo transfer: a review on the optimal endometrial preparation and timing.
      ). As a delay in the detection of peak hormone concentrations was described, it seems reasonable to assume that FET on day 5 after a positive urine LH test might be successful (
      • Cekan S.Z.
      • Beksac M.S.
      • Wang E.
      • Shi S.
      • Masironi B.
      • Landgren B.M.
      • Diczfalusy E.
      The prediction and/or detection of ovulation by means of urinary steroid assays.
      ).
      Monitoring for a spontaneous LH surge using serum analysis can burden the women with repeated monitoring visits (
      • Fatemi H.M.
      • Kyrou D.
      • Bourgain C.
      • Van den Abbeel E.
      • Griesinger G.
      • Devroey P.
      Cryopreserved-thawed human embryo transfer: spontaneous natural cycle is superior to human chorionic gonadotropin-induced natural cycle.
      ;
      • Weissman A.
      • Horowitz E.
      • Ravhon A.
      • Steinfeld Z.
      • Mutzafi R.
      • Golan A.
      • Levran D.
      Spontaneous ovulation versus HCG triggering for timing natural-cycle frozen-thawed embryo transfer: a randomized study.
      ). An alternative to venepuncture is a urine LH test, an inexpensive, easy-to use option, making the timing of FET patient-friendly. A urine LH test is positive 12–36 h after the plasma LH surge (
      • Martinez F.
      • Trounson A.
      • Besanko M.
      Detection of the LH surge for AID, AIH and embryo transfer using a twice daily urinary dip-stick assay.
      ). The challenge with urine LH tests is that the ovulation time cannot be precisely determined because of the high physiological variability; hence, the beginning of the WOI cannot be precisely determined. However, the outcomes were not significantly different with FET performed over the range of 3 days. Ovulation sets in motion processes leading to a period of optimal endometrial receptivity (
      • Hoff J.D.
      • Quigley M.E.
      • Yen S.S.
      Hormonal dynamics at midcycle: a reevaluation.
      ). Associations were identified between clinical pregnancy and various endometrial receptivity markers, indicating their poor ability to predict pregnancy (
      • Craciunas L.
      • Gallos I.
      • Chu J.
      • Bourne T.
      • Quenby S.
      • Brosens J.J.
      • Coomarasamy A.
      Conventional and modern markers of endometrial receptivity: a systematic review and meta-analysis.
      ). Considering similar ultrasonographic endometrial characteristics and results obtained regardless of the day of FET, there may be some degree of individual variability in WOI length. Consequently, endometrial receptivity tests are likely to be relevant only for women with recurrent implantation failure.
      In this study, FET was planned solely based on the day of the urine LH surge without sonographic evidence of ovulation, which may occur up until the second morning after the detection of the urine LH surge (
      • Pearlstone A.C.
      • Surrey E.S.
      The temporal relation between the urine LH surge and sonographic evidence of ovulation: determinants and clinical significance.
      ). When comparing the FET cycle outcomes during embryo transfer on days 5, 6 and 7, no differences in clinical pregnancy rate, implantation rate, miscarriage rate or live birth rate were observed among the groups when adjusted for age, number and quality of transferred blastocysts. Bartels and co-workers also found equally good results with day 6 and day 7 FET, although there was some decline on day 7, consistent with the observations of the current study (
      • Bartels C.B.
      • Ditrio L.
      • Grow D.R.
      • O'Sullivan D.M.
      • Benadiva C.A.
      • Engmann L.
      • Nulsen J.C.
      The window is wide: flexible timing for vitrified-wormed embryo transfer in natural cycle.
      ). This might be a coincidence due to the low number of transferred blastocysts or the decline may suggest that the WOI could be closing.
      The main limitation of the current study is the nature of its design. It is a single-centre retrospective analysis, and despite an acceptable number of included cycles and robust methodological approaches, the presence of potential selection bias could not be completely excluded. Systematic differences among the three groups, not captured in cycle characteristics analysis, may exist and not be evident because the cycles and not the women were analysed. In this study the groups consisted of non-homogeneous participants, including those with single- and double-embryo transfers and a wide array of blastocyst quality. This issue was addressed using propensity score matching, aiming to eliminate potential bias. A properly designed prospective randomized study is needed to confirm the findings.
      Another limitation of this study is that several FET cycles were performed for the same couple. Since the study consisted of neither independent nor repeated measures, specific statistical methods were used to counteract the issue. The Cochran–Mantel–Haenszel test compared factorial variables among different groups while considering the successive number of embryo transfers in the respective couple. As no appropriate method exists for continuous variables in the matter, the respective generalized linear mixed model was built for each variable using the couple's unique ID as a random blocking variable. In this way, the potential bias of repeated cycles in the same couple was eliminated as best as possible. Again, a prospective, randomized study is needed to confirm the findings.

      Conclusion

      The results of this study suggest that vitrified-warmed blastocysts can be transferred into a tNC on day 5, 6 or 7 based on the positive LH urine test without impacting the clinical outcome.

      Acknowledgements

      The study was part of the research programme P3–0327 funded by the Slovenian Research Agency.

      References

        • Bartels C.B.
        • Ditrio L.
        • Grow D.R.
        • O'Sullivan D.M.
        • Benadiva C.A.
        • Engmann L.
        • Nulsen J.C.
        The window is wide: flexible timing for vitrified-wormed embryo transfer in natural cycle.
        Reprod. Biomed. Online. 2019; 39: 241-248
        • Casper R.F.
        • Yanushpolsky E.H.
        Optimal endometrial preparation for frozen embryo transfer cycles: window of implantation and progesterone support.
        Fertil. Steril. 2016; 105 (https://doi.org/): 867-872https://doi.org/10.1016/j.fertnstert.2016.01.006
        • Cekan S.Z.
        • Beksac M.S.
        • Wang E.
        • Shi S.
        • Masironi B.
        • Landgren B.M.
        • Diczfalusy E.
        The prediction and/or detection of ovulation by means of urinary steroid assays.
        Contraception. 1986; 33 (https://doi.org/): 327-345https://doi.org/10.1016/0010-7824(86)90095-8
        • Craciunas L.
        • Gallos I.
        • Chu J.
        • Bourne T.
        • Quenby S.
        • Brosens J.J.
        • Coomarasamy A.
        Conventional and modern markers of endometrial receptivity: a systematic review and meta-analysis.
        Hum. Reprod. Update. 2019; 1 (252): 202-223
        • Direito A.
        • Bailly S.
        • Mariani A.
        • Ecochard R.
        Relationships between the luteinizing hormone surge and other characteristics of the menstrual cycle in normally ovulating women.
        Fertil. Steril. 2013; 99 (e3https://doi.org/): 279-285https://doi.org/10.1016/j.fertnstert.2012.08.047
        • Fatemi H.M.
        • Kyrou D.
        • Bourgain C.
        • Van den Abbeel E.
        • Griesinger G.
        • Devroey P.
        Cryopreserved-thawed human embryo transfer: spontaneous natural cycle is superior to human chorionic gonadotropin-induced natural cycle.
        Fertil. Steril. 2010; 94 (https://doi.org/): 2054-2058https://doi.org/10.1016/j.fertnstert.2009.11.036
        • Glass R.H.
        • Speroff L.
        Clinical gynecologic endocrinology and infertility.
        Fifth Edition. Williams & Wilkins, Baltimore1994
        • Greco E.
        • Litwicka K.
        • Arrivi C.
        • Varricchio M.T.
        • Caragia A.
        • Greco A.
        • Minasi M.G.
        • Fiorentino F.
        The endometrial preparation for frozen-thawed euploid blastocyst transfer: a prospective randomized trial comparing clinical results from natural modified cycle and exogenous hormone stimulation with GnRH agonist.
        J. Assist. Reprod. Genet. 2016; 33 (https://doi.org/): 873-884https://doi.org/10.1007/s10815-016-0736-y
        • Groenewoud E.R.
        • Cantineau A.E.P.
        • Kollen B.J.
        • Macklon N.S.
        • Cohlen B.J.
        What is the optimal means of preparing the endometrium in frozen-thawed embryo transfer cycles? A systematic review and meta-analysis.
        Hum. Reprod. Update. 2013; 19 (https://doi.org/): 458-470https://doi.org/10.1093/humupd/dmt030
        • Groenewoud E.R.
        • Cohlen B.J.
        • Al-Oraiby A.
        • Brinkhuis E.A.
        • Broekmans F.J.M.
        • de Bruin J.P.
        • van den Dool G.
        • Fleisher K.
        • Friederich J.
        • Goddijn M.
        • Hoek A.
        • Hoozemans D.A.
        • Kaaijk E.M.
        • Koks C.A.M.
        • Laven J.S.E.
        • van der Linden P.J.Q.
        • Manger A.P.
        • Slappendel E.
        • Spinder T.
        • Kollen B.J.
        • Macklon N.S.
        A randomized controlled, non-inferiority trial of modified natural versus artificial cycle for cryo-thawed embryo transfer.
        Hum. Reprod. 2016; 31 (https://doi.org/): 1483-1492https://doi.org/10.1093/humrep/dew120
        • Groenewoud E.R.
        • Kollen B.J.
        • Macklon N.S.
        • Cohlen B.J.
        Spontaneous LH surges prior to HCG administration in unstimulated-cycle frozen-thawed embryo transfer do not influence pregnancy rates.
        Reprod. Biomed. Online. 2012; 24 (https://doi.org/): 191-196https://doi.org/10.1016/j.rbmo.2011.11.003
        • Hoff J.D.
        • Quigley M.E.
        • Yen S.S.
        Hormonal dynamics at midcycle: a reevaluation.
        J. Clin. Endocrinol. Metab. 1983; 57 (https://doi.org/): 792-796https://doi.org/10.1210/jcem-57-4-792
        • Huberlant S.
        • Vaast M.
        • Anahory T.
        • Tailland M.L.
        • Rougier N.
        • Ranisavljevic N.
        • Hamamah S.
        Natural cycle for frozen-thawed embryo transfer: Spontaneous ovulation or triggering by HCG.
        Gynecol. Obstet. Fertil. Senol. 2018; 46 (https://doi.org/): 466-473https://doi.org/10.1016/j.gofs.2018.03.006
        • Humaidan P.
        • Kol S.
        • Papanikolaou E.G.
        GnRH agonist for triggering of final oocyte maturation: time for a change of practice?.
        Hum. Reprod. Update. 2011; 17 (https://doi.org/): 510-524https://doi.org/10.1093/humupd/dmr008
        • Kovacic B.
        • Vlaisavljevic V.
        • Reljic M.
        • Cizek-Sajko M.
        Developmental capacity of different morphological types of day 5 human morulae and blastocysts.
        Reprod. Biomed. Online. 2004; 8 (https://doi.org/): 687-694https://doi.org/10.1016/s1472-6483(10)61650-1
        • Kovačič B, V.V.
        Importance of blastocyst morphology in selection for transfer.
        Advances in Embryo Transfer. Intech, Rijeka2012: 161-177
        • Kyrou D.
        • Kolibianakis E.M.
        • Fatemi H.M.
        • Grimbizis G.F.
        • Theodoridis T.D.
        • Camus M.
        • Tournaye H.
        • Tarlatzis B.C.
        • Devroey P.
        Spontaneous triggering of ovulation versus HCG administration in patients undergoing IUI: a prospective randomized study.
        Reprod. Biomed. Online. 2012; 25 (https://doi.org/): 278-283https://doi.org/10.1016/j.rbmo.2012.05.005
        • Lessey B.A.
        • Young S.L.
        What exactly is endometrial receptivity?.
        Fertil. Steril. 2019; 111 (https://doi.org/): 611-617https://doi.org/10.1016/j.fertnstert.2019.02.009
        • Mackens S.
        • Santos-Ribeiro S.
        • van de Vijver A.
        • Racca A.
        • Van Landuyt L.
        • Tournaye H.
        • Blockeel C.
        Frozen embryo transfer: a review on the optimal endometrial preparation and timing.
        Hum. Reprod. 2017; 32 (https://doi.org/): 2234-2242https://doi.org/10.1093/humrep/dex285
        • Mackens S.
        • Stubbe A.
        • Santos-Ribeiro S.
        • et al.
        To trigger or not to trigger ovulation in a natural cycle for frozen embryo transfer: a randomized controlled trial.
        Hum. Reprod. 2020; 35: 1073-1081
        • Martinez F.
        • Trounson A.
        • Besanko M.
        Detection of the LH surge for AID, AIH and embryo transfer using a twice daily urinary dip-stick assay.
        Clin. Reprod. Fertil. 1986; 4: 45-53
        • Navot D.
        • Bergh P.A.
        • Williams M.
        • Garrisi G.J.
        • Guzman I.
        • Sandler B.
        • Fox J.
        • Schreiner-Engel P.
        • Hofmann G.E.
        • Grunfeld L.
        An insight into early reproductive processes through the in vivo model of ovum donation.
        J. Clin. Endocrinol. Metab. 1991; 72 (https://doi.org/): 408-414https://doi.org/10.1210/jcem-72-2-408
        • Park S.J.
        • Goldsmith L.T.
        • Skurnick J.H.
        • Wojtczuk A.
        • Weiss G.
        Characteristics of the urinary luteinizing hormone surge in young ovulatory women.
        Fertil. Steril. 2007; 88 (https://doi.org/): 684-690https://doi.org/10.1016/j.fertnstert.2007.01.045
        • Pearlstone A.C.
        • Surrey E.S.
        The temporal relation between the urine LH surge and sonographic evidence of ovulation: determinants and clinical significance.
        Obstet. Gynecol. 1994; 83: 184-188
        • Psychoyos A.
        Uterine receptivity for nidation.
        Ann. N Y Acad. Sci. 1986; 476 (https://doi.org/): 36-42https://doi.org/10.1111/j.1749-6632.1986.tb20920.x
        • Reljič M.
        • Knez J.
        Predicted luteal phase length has no influence on success of vitrified-warmed blastocyst transfer in natural cycle.
        J. Ovarian Res. 2018; 11 (https://doi.org/): 63https://doi.org/10.1186/s13048-018-0436-6
        • Su H.W.
        • Yi Y.C.
        • Wei Ty Chang T.C.
        • Cheng C.M.
        Detection of ovulation,a review of currently available methods.
        Bioeng Transl. Med. 2017; 2: 238-246https://doi.org/10.1002/btm2.10058
        • Weissman A.
        • Horowitz E.
        • Ravhon A.
        • Steinfeld Z.
        • Mutzafi R.
        • Golan A.
        • Levran D.
        Spontaneous ovulation versus HCG triggering for timing natural-cycle frozen-thawed embryo transfer: a randomized study.
        Reprod. Biomed. Online. 2011; 23 (https://doi.org/): 484-489https://doi.org/10.1016/j.rbmo.2011.06.004

      Biography

      Vida Gavrić Lovrec, MD, PhD, was awarded a PhD by the University of Ljubljana in the field of perifollicular blood flow characteristics in IVF. She is involved in several research projects covering different fields of IVF at the Department of Reproductive Medicine and Gynecologic Endocrinology, University Medical Centre Maribor, Slovenia.
      Key message
      The transfer of vitrified-warmed blastocysts may be scheduled on day 5, 6 or 7 after the LH surge as detected by urine tests without significantly affecting either clinical pregnancy rate or live birth rate.