The effectiveness of vitrified zygote accumulation for managing the extremely diminished ovarian reserve in infertile women: a retrospective cohort study

The effectiveness of vitrified zygote accumulation for managing the extremely diminished ovarian reserve in infertile women: a retrospective cohort study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The effectiveness of vitrified zygote accumulation for managing the extremely diminished ovarian reserve in infertile women: a retrospective cohort study Pi-Tsang Huang, Ni-Chin Tsai, Yu-Ju Lin, Hao-Ting Lien, Yu-Yang Hsiao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4573045/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Accumulated vitrified embryos are stored for later thawed transfer to deal with poor responders. However, the cost and outcome of vitrified zygotes have yet to be studied. Our study aimed to investigate the effectiveness of the vitrified zygote accumulation method in diminished ovarian reserve patients. Methods This retrospective study included 122 diminished ovarian reserve patients who met the Poseidon classification groups 3 and 4 from June 1, 2019, to December 31, 2022. The patients underwent controlled ovarian stimulation and embryo transfer using accumulated vitrified zygotes. The clinical pregnancy rate and live birth rate per embryo transfer and stimulation cycle are evaluated. Results Two hundred seventy-eight IVF/ICSI cycles in one hundred twenty-two females treated at our institution were divided into two groups, according to age < 40 years (n = 51) and ≥ 40 years (n = 71). The younger group had significantly more favorable outcomes than the older group only in live birth rates per cycle (12.6% vs. 4%, p = 0.029) and per transfer (24.1% vs. 9.9%, p = 0.027). However, the older group had more embryos per transfer (2.4 ± 0.9 vs. 2.0 ± 0.8, p = 0.014). There were no differences between the number of stimulation cycles per patient, the number of oocytes and mature oocytes retrieved, the number of transfer cycles per patient, the cost per patient, or transfer. We set a model mimicking the cost of fresh embryos. The zygote accumulation strategy costs were higher per patient and per cycle than those of the fresh embryo transfer model. Conclusions In diminished ovarian reserve women undergoing IVF/ICSI treatment, younger women had significantly better live birth rates than the older women applying the vitrified zygote accumulation strategy, even though the older group had a higher number of embryos per transfer. However, the zygote accumulation strategy benefits older women more than fresh embryo transfer. The strategy might cost more but decrease the anxiety and stress. Diminished ovarian reserve (DOR) Embryo transfer Vitrification Zygote accumulation Figures Figure 1 Introduction Diminished or poor ovarian reserve (DOR/POR) is a challenge that infertility couples and physicians may face during in vitro fertilization (IVF) treatment. The Bologna criteria, published by the European Society of Human Reproduction and Embryology in 2011, set cutoff values for age, antral follicle count (AFC), anti-mullerian hormone (AMH), and response to stimulation in a previous cycle as indicators of DOR/POR[1]. The POSEIDON (Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number) criteria, a more recent classification system, further subdivides predicted poor responders into four groups based on age, ovarian reserve markers, and prior inadequate response [2]. DOR/POR patients face significant challenges in their IVF journey. They may encounter less oocyte retrieval, a lack of good-quality embryos during transfer, and a higher drop-out rate[3]. These difficulties result in lower live birth rates than normal responders[4–6]. Despite the numerous interventions or modifications of the treatment protocol published by previous studies, none have been proven advantageous[7–10]. This underscores the need for further research and potential solutions. Cryopreserved oocytes offer a promising solution for DOR/POR patients[11–14]. Published data suggests that accumulating oocytes or embryos from consecutive stimulation cycles may be beneficial[15–17]. The concept is simple yet powerful: collecting oocytes with multiple ovarian stimulation cycles helps increase the chances of live birth by mimicking a 'normal responder-like' status. Vitrification, the best cryopreservation method for all developmental stages[18], has shown significant potential. A previous study reported that oocyte accumulation by vitrification significantly lessens DOR/POR patients' drop-out and IVF cancellation rates[19]. However, the live birth rate remains a topic of debate among studies[19, 20], indicating the need for further investigation and refinement of this strategy. Datta et al.[21] demonstrated that the accumulation of embryos may increase the chance of better selection and more embryos available for transfer. The strategy increases the birth rate and fewer cycle cancellations in DOR/POR patients. Nonetheless, Ge et al. found that embryo banking in women undergoing IVF with POR according to the Bologna criteria decreases the cumulative live birth rate and prolongs the time to live birth. [22]. Currently, the outcome of embryo accumulation following multiple ovarian stimulations in DOR/POR patients is still contradictory. After thawing, the pronuclear stage cryopreservation shows a survival rate of over 90% because of its single-cell form, absence of spindle apparatus, and easy diagnosable survival through syngamy passage and progression to first cleavage [23]. Shapiro et al., in a randomized control trial, compared the clinical outcomes of freeze-all stemming from zygote or blastocyst cryopreservation, and found the two methods to be equally effective [24]. However, the outcome and cost of vitrified zygotes have not been studied yet. Therefore, this study aims to fill this gap by investigating the effectiveness of the vitrified zygote accumulation method in DOR/POR patients, offering a potentially intriguing avenue for further research. Materials and methods Participants This is a retrospective cohort study that examined the medical records of women with diminished ovarian reserve (DOR) or poor ovarian response (POR) who underwent controlled ovarian stimulation (COS) and embryo transfer (ET) using vitrified embryos. The study occurred at our institution from June 1, 2019, to December 31, 2022. DOR was defined as having a low antral follicle count (AFC < 5) or a low serum anti-Müllerian hormone (AMH) level (< 1.2 ng/ml) at the start of ovarian stimulation. The study included women in Poseidon Groups 3 and 4 with at least one vitrified zygote with two pronuclei (2PN) created for intended transfer after multiple stimulation cycles. Exclusion criteria were the use of donor oocytes, preimplantation genetic testing (PGT), and failure to retrieve oocytes after COS. All intracytoplasmic sperm injection (ICSI) was performed due to male factors. The study evaluated patient characteristics such as age, body mass index, family history, primary or secondary infertility, cause of infertility, and AMH levels. The decision to stop zygote accumulation was made through a discussion between the patient and the physician. The Institutional Review Board and the Chang Gung Memorial Hospital Ethics Committee in Kaohsiung, Taiwan, approved the study (IRB No.: 202400767B0). Protocols The ovarian stimulation protocols included gonadotropin-releasing hormone (GnRH) antagonist protocols and progestin-primed ovarian stimulation protocols, all performed following standard clinical practice. Each protocol was selected based on factors such as ovarian reserve, age, and previous ovarian response. The dose of gonadotropin was adjusted based on the individual response. After the maturation of follicles, oocytes were retrieved, and standard IVF or ICSI procedures were used for fertilization, as previously described[25, 26]. The presence of clear 2PNs was confirmed prior to the start of vitrification to avoid vitrifying zygotes in syngamy. The details of oocyte grading assessment of fertilization, embryo culture, zygote, embryo grading, endometrial preparation for frozen embryo transfer are described elsewhere [27]. The choice between Day 3 embryo transfer and extended culture to blastocyst and transfer on Day 5 was based on embryo quality and number[28]. Vitrification The method of zygote vitrification/warming was described by Kuwayama et al. [29]. We used the Cryotech Vitrification/warming kit according to the manufacturer’s protocols. Transfer the Zygote on the surface of ES (Equilibration Solution) for 12-15min; after complete equilibration, transfer the Zygote to the VS (Vitrification Solution) for 60-90sec, then place the Zygote near the black mark on the Cryotec sheet with small volume, immediately submerge and stir the Cryotec in fresh liquid nitrogen. Warming Quickly put the Cryotec from liquid nitrogen into the TS for l min, then aspirate the Zygote to the bottom of the DS (Dilution solution) well for 3 min. Wash the Zygote to WS1(Washing Solution ) for 5 min, then WS2 for 1 min. Then, culture the zygote in Universal IVF Medium (ORIGIO) for 1hr then transfer to the sequential medium G1/G 2 (Vitrolife) media until cleavage stage or blastocyst stage. Costs The costs were based on the actual cost for each patient, as recorded in their clinical records. The oocyte pickup (OPU) cost included charges for procedures regarding oocyte retrieval, pre-op tests, anesthesia, insemination, and embryo culture costs. The costs for the hormonal test and sonography included the test on the FSH, estradiol, progesterone, LH, and sonography during the COH cycle. Costs for COH medication included drugs for COH and the triggering of ovulation. Costs of embryo transfer, including vitrification of zygote, embryo thawing, medication for endometrium priming, monitoring with sonography, hormonal tests, embryo transfer, and luteal phase support after ET, all of which had accumulated until the day of the urinary hCG test 2 weeks after embryo transfer. All charges were calculated in Taiwanese Dollars (NTD). Outcome measures The study focused on outcome measures, with the primary outcome being clinical pregnancy, defined as the presence of gestational sac(s) on transvaginal ultrasound after 6–7 weeks of gestation. Clinical abortion was recorded in cases of pregnancy loss between the clinical detection of pregnancy and the 22nd week of gestation. Statistical analysis Statistical analysis was conducted using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as mean and standard deviation and compared using Mann–Whitney U tests. Categorical variables were expressed as proportion and percentages and compared using Fisher's exact test. The effects of potential clinical and laboratory factors were examined, and the statistical significance of each variable was evaluated using univariate analysis. A p -value of < 0.05 was considered statistically significant in all analyses Results We have reviewed the medical records of 278 IVF/ICSI cycles conducted on 122 women at our institution over a 2-year period. Table 1 shows the characteristics of these patients and the types of assisted reproduction techniques used. The average age of the patients was 40.0 ± 3.5 years, and the mean serum AMH level was 0.78 ± 0.5 ng/mL. Of the cycles, 22 (7.9%) resulted in no oocytes being retrieved, while 256 (92.1%) had at least one oocyte retrieved for culture. Only one woman (0.8%) did not have an embryo for transfer because she stopped the treatment protocol after a failed oocyte retrieval stimulation. The clinical pregnancy rate was 22.3% per transfer and 11.2% per cycle (Table 2). We divided all cycles into two groups based on maternal age using the Poseidon classification: those where the female was < 40 years (n = 51) and those where the female was ≥ 40 years (n = 71) (Table 3). Our results demonstrated that the younger group had significantly more favorable outcomes compared to the older group, particularly in live birth rates per cycle (12.6% vs. 4%, p = 0.029) and per transfer (24.1% vs. 9.9%, p = 0.027). However, the older group had more embryos per transfer (2.4 ± 0.9 vs. 2.0 ± 0.8, p = 0.014). There were no differences between the number of stimulation cycles per patient, the number of oocytes and mature oocytes retrieved, the number of transfer cycles per patient, the cost per patient, or per transfer. The younger group seemed to have a better clinical pregnancy rate per cycle (15.5% vs. 8.4%, p = 0.066) and per transfer (29.6% vs. 21.1%, p = 0.088), but both showed no significance. Table 4 shows the costs of zygote accumulations. We set a model mimicking the cost of fresh embryo transfer with the same number of stimulation and embryo transfer cycles. The costs of the zygote accumulation strategy were higher both per patient (NTD 257,164 vs. 161,508) and per cycle (NTD 225,066 vs. 141,755) than those of the fresh embryo transfer model. Figure 1 shows the difference (maximum minus the minimum number) of oocyte retrieval and pronuclei (2PN) formation in the same patient treated with different ovarian stimulation. 82.8% (101/122) and 90%(112/125) of patients’ differences in oocyte retrieval and pronuclei (2PN) formation varied between 0 and 2. Discussion This retrospective study, we found that younger women undergoing vitrified zygote accumulation strategy with diminished ovarian reserve had significantly better live birth rates than the older women, even though the older group had a higher number of embryos per transfer. The zygote accumulation strategy might cost more than fresh embryo transfer if the number of stimulations and embryo transfer cycles are the same in a patient. The main difference in the cost includes vitrification of the zygote, embryo thawing, medication for endometrium preparation, monitoring with sonography, and hormonal tests. Patients included in our study were poor responders (median AMH = 0.78 ng/mL) with advanced age (median age = 40), and these patients are challenging for physicians to deal with. Our study is one of the few to deal with such low ovarian reserve and older women undergoing repeated zygote freezing with autologous oocytes from IVF/ICSI. Besides, this is the first study to report the outcome and costs of the zygote accumulation strategy. The outcome and the price may represent the effectiveness of zygote accumulation in women with extremely diminished ovarian reserve. Su et al. [27],, in a previous study in our institution, investigated females from whom zero, one, two, or three oocytes were retrieved following fresh IVF/ICSI. All cycles were divided according to maternal age using the Bologna criteria into those in which the female was < 40 years (n = 592) and ≥ 40 years (n = 266). The results demonstrated that the younger group had significantly more favorable clinical pregnancy rates than the older group. Our study also divided women into < 40 and ≥ 40 years old (Table 5), and we had a similar average age in both groups. Besides, in the younger group (< 40 years old) compared to a previous study, we reported lower clinical pregnancy rate per cycle (15.5%[16/103] vs. 20.7%[123/592]) and live birth rate per cycle (12.6%[13/103] vs. 17.2%[102/592]), but better clinical pregnancy rate per transfer (29.6%[16/54] vs. 27.7%[123/444]) and live birth rate per transfer (24.1%[13/54] vs. 23%[102/444]). On the other hand, our study demonstrated better outcomes in the advanced age group with clinical pregnancy rate per transfer (21.1%[15/71] vs. 7.6%[14/185]), clinical pregnancy rate per cycle (8.4%[15/175] vs. 5.3%[14/266]), live birth rate per transfer (9.9%[7/71] vs. 5.4% [10/185]) and live birth rate per cycle(4% [7/175] vs. 3.8% [10/266]). These results may indicate that zygote accumulation strategy should be considered in older females undergoing IVF/ICSI. In a study by Datta et al. [21], the results of transferring frozen embryos collected over three consecutive modified natural cycles in women with low ovarian reserve were examined. It was found that the live birth rate after a single embryo transfer following three consecutive modified natural cycles was significantly higher compared to a fresh embryo transfer in a single modified natural IVF cycle (30.6% vs. 13.3% respectively; p = 0.002). Additionally, the cumulative live birth rate for all embryo transfers was also notably higher in the accumulation group. However, it's important to note that there may be some bias in this comparison, as it was based on all transfers from embryo accumulation over three stimulation cycles versus a fresh embryo transfer after one stimulation cycle. On the other hand, based on the Bologna criteria, Ge et al. implemented a different approach to collecting embryos from women with a poor response undergoing IVF/ICSI treatment. In their study, all couples were given multiple stimulation cycles until they had at least five embryos for transfer. The findings revealed that embryo banking in women with poor ovarian response led to a significantly lower cumulative live birth rate and a longer time to achieve live birth compared to not using embryo banking. The conclusion drawn was that employing an embryo banking strategy for poor responders is less effective than a conventional non-banking approach[22]. Our study's selection of the POSEIDON criteria differed from those used in the previously mentioned two studies, Datta et al. [21] and Ge et al. [22], which adopted the Bologna criteria. Moreover, the methods of embryo accumulation were different compared with the two studies. Datta et al. transferred accumulated frozen embryos after three consecutive modified natural cycles in women with low ovarian reserve, regardless of the number of embryos obtained. On the other hand, Ge et al. provided several stimulation cycles until at least five embryos were available for transfer, so every participant had a chance for embryo transfer. The average stimulation cycle per woman was 5.3 to accumulate five embryos available for transfer. In comparison, we vitrified the zygotes rather than the embryos. In addition, we didn't set a goal for the number of stimulation cycles or the number of embryos available for transfer. We let the patient and the physician decide when to stop the zygote accumulation program according to the physician's expertise and the patient's financial, physical, and mental conditions. It was more representative to reflect routine clinical practice in which poor responders may be present. It is reasonable that the dropout rate and the number of patients without embryos for transfer were low when the embryo and zygote accumulation strategy were applied. In the Datta et al. study, nine patients (7.2%) had no embryos for transfer with zero dropout incidence[21]. On the other hand, every patient in the banking group completed the study and had a transferable embryo, as shown in Ge et al.’s report[22]. In our study, only one woman (0.8%) had no embryo for transfer and dropped out, with the average stimulation cycles per woman being 2.3. Infertile couples experience pressure from society, family, and treatment and have a higher risk of negative emotions, such as anxiety and depression [30–32]. Stress and anxiety levels remained elevated across all cycles. Women with lower stress and anxiety levels on the day before oocyte retrieval had a higher pregnancy rate[33]. Since the average stimulation cycles per patient were 2.3 in our study and the couples don’t need to confront the outcome of embryo transfer as much as fresh transfer, we might presume that the stress and anxiety levels were lower with the zygote accumulation strategy. Also, it might contribute to the low dropout rate. After controlled ovarian stimulation, we also reported the variation in oocyte and pronuclei (2PN) yield. More than 80 percent of patients’ differences (maximum minus the minimum number) in oocyte retrieval and pronuclei (2PN) formation varied between 0 and 2 (Fig. 1). This result implies that a patient may fluctuate in follicle recruitment during different menstrual cycles. Researchers Rustamov et al. discovered that, even with personalized treatment, there is a limit to how much variation in response can be minimized[34]. They found that there could be a 3.4-fold difference in yield between two treatment cycles for patients with similar characteristics. Similarly, when the same patient was treated in the same way on two occasions, a 2.7-fold difference in yield could be observed. These findings have significant clinical implications. For instance, if a patient obtained nine oocytes from a cycle, it can be expected that another patient with similar characteristics and similar treatment would produce between 6 and 13 oocytes. Any response in the range of 4 to 19 oocytes would not be surprising. If the same patient were stimulated in the same way a second time, it could be anticipated that the number of oocytes retrieved would fall between 7 and 12, with an expected range of 5 to 17 oocytes. The limitations of our study are its retrospective design and being performed at a single center, so results may need to be more generalizable to other populations and clinical settings. Also, although we have the same team that has previously published a non-accumulating zygote strategy for similar patients for comparison, we didn’t set a well-designed control group so that the outcome couldn’t be compared to other methods. Randomized controlled trials with adequate sample size should be conducted to confirm these findings. Conclusion In conclusion, by applying the vitrified zygote accumulation strategy in women with a poor response based on the POSEIDON criteria starting their IVF/ICSI treatment, younger women had significantly better live birth rates than the older women, even though the older group had a higher number of embryos per transfer. However, the zygote accumulation strategy benefits older women more than fresh embryo transfer. The strategy might cost more but decrease the anxiety and stress. Abbreviations 2PN: two pronuclei; AFC :Antral follicle count; AMH :Anti‑Müllerian hormone; COS :Controlled ovarian stimulation; DOR: Diminished ovarian reserve; ET: Embryo transfer; ICSI:Intracytoplasmic sperm injection; IVF: In vitro fertilization; GnRH: Gonadotropin‑releasing hormone; PGT: Preimplantation genetic testing; POR :Poor ovarian reserve Declarations Acknowledgements The authors thank the staff of Kaohsiung Chang Gung Memorial Hospital for the tribute and the involvement in this study. Authors’ contributions K‑C L, and P‑T H contributed to the conception and design of the study, collecting patients in Kaohsiung Chang Gung Memorial Hospital and data analysis as well as interpretation. P-T H, N-C T, Y-J L, H-T L, Y-Y H, Y-T S, H-W H, K-C L were involved in discussion and advising the study design. K‑C L was the co‑corresponding author and played the major role in the design of manuscript, interpreting the analyzed data, advising, drafting, and revising the draft. All authors were involved in the writing of the manuscript and provided final approval. Funding : This study was supported by CMRPG8M0241-2 from Chang Gung Memorial Hospital. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The Institutional Review Board and the Chang Gung Memorial Hospital Ethics Committee in Kaohsiung, Taiwan, approved the study (IRB No.: 202400767B0). There is no participants’ consent for this retrospective study and Institutional Review Board decides this is ethically acceptable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Ferraretti, A.P., et al., ESHRE consensus on the definition of 'poor response' to ovarian stimulation for in vitro fertilization: the Bologna criteria. 2011(1460-2350 (Electronic)). Poseidon, G., et al., A new more detailed stratification of low responders to ovarian stimulation: from a poor ovarian response to a low prognosis concept. Fertil Steril, 2016. 105 (6): p. 1452-3. Verberg, M.F., et al., Why do couples drop-out from IVF treatment? A prospective cohort study. 2008(1460-2350 (Electronic)). Drakopoulos, P., et al., Conventional ovarian stimulation and single embryo transfer for IVF/ICSI. How many oocytes do we need to maximize cumulative live birth rates after utilization of all fresh and frozen embryos? 2016(1460-2350 (Electronic)). Sunkara, S.K., et al., Association between the number of eggs and live birth in IVF treatment: an analysis of 400 135 treatment cycles. 2011(1460-2350 (Electronic)). van der Gaast, M.H., et al., Optimum number of oocytes for a successful first IVF treatment cycle. Reprod Biomed Online, 2006(1472-6483 (Print)). Giovanale, V., et al., Poor responders in IVF: an update in therapy. Gynecological Endocrinology, 2015(1473-0766 (Electronic)). Haahr, T., et al., Management Strategies for POSEIDON Groups 3 and 4. Front Endocrinol (Lausanne), 2019. 10 : p. 614. Jin, B., et al., Comparison of clinical outcomes among dual ovarian stimulation, mild stimulation and luteal phase stimulation protocols in women with poor ovarian response. Gynecological Endocrinology, 2018(1473-0766 (Electronic)). Vaiarelli, A., et al., What is new in the management of poor ovarian response in IVF? Current opinion in obstetrics & gynecology, 2018(1473-656X (Electronic)). Cobo, A., et al., Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertility and sterility, 2008(1556-5653 (Electronic)). Cobo, A., et al., Use of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial. Human reproduction, 2010(1460-2350 (Electronic)). Parmegiani, L., et al., Efficiency of aseptic open vitrification and hermetical cryostorage of human oocytes. Reproductive biomedicine online, 2011(1472-6491 (Electronic)). Rienzi, L., et al., Embryo development of fresh 'versus' vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study. Human reproduction, 2010(1460-2350 (Electronic)). Almodin, C.G., et al., Embryo development and gestation using fresh and vitrified oocytes. Human reproduction, 2010(1460-2350 (Electronic)). Domingues, T.S., et al., Egg donation of vitrified oocytes bank produces similar pregnancy rates by blastocyst transfer when compared to fresh cycle. Journal of assisted reproduction and genetics, 2017(1573-7330 (Electronic)). Doyle, J.O., et al., Successful elective and medically indicated oocyte vitrification and warming for autologous in vitro fertilization, with predicted birth probabilities for fertility preservation according to number of cryopreserved oocytes and age at retrieval. Fertility and sterility, 2016(1556-5653 (Electronic)). Rienzi, L., et al., Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. 2017(1460-2369 (Electronic)). Cobo, A., et al., Accumulation of oocytes: a new strategy for managing low-responder patients. Reprod Biomed Online, 2012. 24 (4): p. 424-32. Lee, K.S., et al., The live birth rate of vitrified oocyte accumulation for managing diminished ovarian reserve: a retrospective cohort study. J Ovarian Res, 2023. 16 (1): p. 49. Datta, A.K., et al., Accumulation of embryos over 3 natural modified IVF (ICSI) cycles followed by transfer to improve the outcome of poor responders. Facts Views Vis Obgyn, 2019. 11 (1): p. 77-84. Ge, Q.L., et al., The impact of the embryo banking on the cumulative live birth rate in women with poor ovarian response according to the Bologna criteria. Reprod Med Biol, 2023. 22 (1): p. e12533. Golakov, M.A.-O., et al., What is the net effect of introducing vitrification for cryopreservation of surplus 2PN oocytes in an IVF program? 2018(1432-0711 (Electronic)). Shapiro, B.S., et al., Freeze-all at the blastocyst or bipronuclear stage: a randomized clinical trial. 2015(1556-5653 (Electronic)). Tsai, N.C., et al., Monopronucleated (1PN) and tripronuclear (3PN) zygotes formation during assisted reproduction in POSEIDON group 4 patients: Emphasizing on polar bodies. 2021(1447-0756 (Electronic)). Tsai, N.C., et al., Developmental potential of surplus morulas with delayed and/or incomplete compaction after freezing-thawing procedures. Reprod Biol Endocrinol, 2019. 17 (1): p. 87. Su, Y.T., et al., Age is a major prognosticator in extremely low oocyte retrieval cycles. Taiwan J Obstet Gynecol, 2017. 56 (2): p. 175-180. Lan, K.C., et al., The predictive value of using a combined Z-score and day 3 embryo morphology score in the assessment of embryo survival on day 5. Hum Reprod, 2003. 18 (6): p. 1299-306. Kuwayama, M., Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology, 2007. 67 (1): p. 73-80. Beukers, F., et al., Parental psychological distress and anxiety after a successful IVF/ICSI procedure with and without preimplantation genetic screening: follow-up of a randomised controlled trial. 2012(1872-6232 (Electronic)). LoGiudice, J.A. and J. Massaro, The impact of complementary therapies on psychosocial factors in women undergoing in vitro fertilization (IVF): A systematic literature review. 2017(1532-8201 (Electronic)). Haimovici, F.A.-O., et al., Stress, anxiety, and depression of both partners in infertile couples are associated with cytokine levels and adverse IVF outcome. 2018(1600-0897 (Electronic)). Turner, K., et al., Stress and anxiety scores in first and repeat IVF cycles: a pilot study. PLoS One, 2013. 8 (5): p. e63743. Rustamov, O., et al., How much variation in oocyte yield after controlled ovarian stimulation can be explained? A multilevel modelling study. 2017(2399-3529 (Electronic)). Tables Tables 1-5 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1Demographiccharacteristics.docx Table2Resultsofoocyteretrieval.docx Table3Outcomesofyoungerandolder.docx Table4Thecostsofzygoteaccumulation.docx Table5ComparisonwithSuetal..docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4573045","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":319858454,"identity":"6aaf3155-315b-487c-bbe0-d9b3452c3037","order_by":0,"name":"Pi-Tsang Huang","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Pi-Tsang","middleName":"","lastName":"Huang","suffix":""},{"id":319858455,"identity":"dff2d539-f8d5-4d82-bfb0-7f5be04745e4","order_by":1,"name":"Ni-Chin Tsai","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ni-Chin","middleName":"","lastName":"Tsai","suffix":""},{"id":319858457,"identity":"c40635a8-710c-4150-a2a4-32ef3684de8c","order_by":2,"name":"Yu-Ju Lin","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yu-Ju","middleName":"","lastName":"Lin","suffix":""},{"id":319858458,"identity":"e84b4e77-fb44-42a3-bdd2-06eea4fe07a2","order_by":3,"name":"Hao-Ting Lien","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hao-Ting","middleName":"","lastName":"Lien","suffix":""},{"id":319858459,"identity":"a274cdd8-1f51-4217-b316-4c34778bde60","order_by":4,"name":"Yu-Yang Hsiao","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yu-Yang","middleName":"","lastName":"Hsiao","suffix":""},{"id":319858461,"identity":"4d4e8578-8511-441d-b0f6-3b67b99e8aa1","order_by":5,"name":"Yu-Ting Su","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yu-Ting","middleName":"","lastName":"Su","suffix":""},{"id":319858462,"identity":"bff1c4af-fd32-408f-a5ec-52649aa4d1f8","order_by":6,"name":"Hsuan-Wei Huang","email":"","orcid":"","institution":"Jen-Ai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hsuan-Wei","middleName":"","lastName":"Huang","suffix":""},{"id":319858463,"identity":"30939dc1-1e54-4a1e-a901-94bfecca8d80","order_by":7,"name":"Kuo-Chung Lan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACPgYGZhCdAMSMD4AEDx8hLWxIWpgNQFrYSNHCJgEVIaCF/exhY54KuzyD2+3XKr/m2MmwMTA/fHQDnxaevORknjPJxQZ3zpTdlt2WDHQYm7FxDl6H5Rgf5m07kLjhRk7abcltzEAtPGzSeLXwv0FoKZbcVk+EFokc42SIlvRjjB+3HSZGyxtjwzlnkhNn3shhlmbcdpyHjZmAX/j5c4wl3lTYJfbdSH/48ee2ant+9uaHj/FpQQI8Bsw8IJqZOOUgwP6A8QfxqkfBKBgFo2AEAQBzFEPa1teWPQAAAABJRU5ErkJggg==","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Kuo-Chung","middleName":"","lastName":"Lan","suffix":""}],"badges":[],"createdAt":"2024-06-13 02:29:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4573045/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4573045/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60197273,"identity":"01e8984d-55dc-480c-9076-01b36efba107","added_by":"auto","created_at":"2024-07-13 01:55:38","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":144268,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure1thedifferenceofOPUand2PN.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4573045/v1/d795a156ddb2735484e37c79.jpg"},{"id":78614994,"identity":"91e3ef4c-61d1-4b32-9c4f-2a22d8c886f0","added_by":"auto","created_at":"2025-03-16 16:01:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":680732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4573045/v1/53faf70c-fb00-45f7-8ccf-c2a27ef84f54.pdf"},{"id":60196772,"identity":"1c83fdbc-961c-4319-a149-05a40de3853b","added_by":"auto","created_at":"2024-07-13 01:47:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16822,"visible":true,"origin":"","legend":"","description":"","filename":"Table1Demographiccharacteristics.docx","url":"https://assets-eu.researchsquare.com/files/rs-4573045/v1/c5323b1bf98c4d4b3619f737.docx"},{"id":60197271,"identity":"44300c95-d9a5-404d-b84b-d536a3bc3dd0","added_by":"auto","created_at":"2024-07-13 01:55:38","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16357,"visible":true,"origin":"","legend":"","description":"","filename":"Table2Resultsofoocyteretrieval.docx","url":"https://assets-eu.researchsquare.com/files/rs-4573045/v1/59790653b404c10ab858e3b1.docx"},{"id":60196771,"identity":"5e41f1c2-dbef-43f3-992b-e15d63d47a72","added_by":"auto","created_at":"2024-07-13 01:47:38","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17717,"visible":true,"origin":"","legend":"","description":"","filename":"Table3Outcomesofyoungerandolder.docx","url":"https://assets-eu.researchsquare.com/files/rs-4573045/v1/e386656c1e6d0d509fc9254c.docx"},{"id":60196776,"identity":"054b4a1c-60aa-460c-ae2f-93db09f64477","added_by":"auto","created_at":"2024-07-13 01:47:38","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16552,"visible":true,"origin":"","legend":"","description":"","filename":"Table4Thecostsofzygoteaccumulation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4573045/v1/4c946178b968e8320a22b9e5.docx"},{"id":60198042,"identity":"7f470b7a-46f8-44e6-8743-e9a462e3b87a","added_by":"auto","created_at":"2024-07-13 02:03:38","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15324,"visible":true,"origin":"","legend":"","description":"","filename":"Table5ComparisonwithSuetal..docx","url":"https://assets-eu.researchsquare.com/files/rs-4573045/v1/bdfbf3175230170ce06de4ab.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effectiveness of vitrified zygote accumulation for managing the extremely diminished ovarian reserve in infertile women: a retrospective cohort study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiminished or poor ovarian reserve (DOR/POR) is a challenge that infertility couples and physicians may face during in vitro fertilization (IVF) treatment. The Bologna criteria, published by the European Society of Human Reproduction and Embryology in 2011, set cutoff values for age, antral follicle count (AFC), anti-mullerian hormone (AMH), and response to stimulation in a previous cycle as indicators of DOR/POR[1]. The POSEIDON (Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number) criteria, a more recent classification system, further subdivides predicted poor responders into four groups based on age, ovarian reserve markers, and prior inadequate response [2].\u003c/p\u003e \u003cp\u003eDOR/POR patients face significant challenges in their IVF journey. They may encounter less oocyte retrieval, a lack of good-quality embryos during transfer, and a higher drop-out rate[3]. These difficulties result in lower live birth rates than normal responders[4\u0026ndash;6]. Despite the numerous interventions or modifications of the treatment protocol published by previous studies, none have been proven advantageous[7\u0026ndash;10]. This underscores the need for further research and potential solutions.\u003c/p\u003e \u003cp\u003eCryopreserved oocytes offer a promising solution for DOR/POR patients[11\u0026ndash;14]. Published data suggests that accumulating oocytes or embryos from consecutive stimulation cycles may be beneficial[15\u0026ndash;17]. The concept is simple yet powerful: collecting oocytes with multiple ovarian stimulation cycles helps increase the chances of live birth by mimicking a 'normal responder-like' status. Vitrification, the best cryopreservation method for all developmental stages[18], has shown significant potential. A previous study reported that oocyte accumulation by vitrification significantly lessens DOR/POR patients' drop-out and IVF cancellation rates[19]. However, the live birth rate remains a topic of debate among studies[19, 20], indicating the need for further investigation and refinement of this strategy.\u003c/p\u003e \u003cp\u003eDatta et al.[21] demonstrated that the accumulation of embryos may increase the chance of better selection and more embryos available for transfer. The strategy increases the birth rate and fewer cycle cancellations in DOR/POR patients. Nonetheless, Ge et al. found that embryo banking in women undergoing IVF with POR according to the Bologna criteria decreases the cumulative live birth rate and prolongs the time to live birth. [22]. Currently, the outcome of embryo accumulation following multiple ovarian stimulations in DOR/POR patients is still contradictory.\u003c/p\u003e \u003cp\u003eAfter thawing, the pronuclear stage cryopreservation shows a survival rate of over 90% because of its single-cell form, absence of spindle apparatus, and easy diagnosable survival through syngamy passage and progression to first cleavage [23]. Shapiro et al., in a randomized control trial, compared the clinical outcomes of freeze-all stemming from zygote or blastocyst cryopreservation, and found the two methods to be equally effective [24]. However, the outcome and cost of vitrified zygotes have not been studied yet. Therefore, this study aims to fill this gap by investigating the effectiveness of the vitrified zygote accumulation method in DOR/POR patients, offering a potentially intriguing avenue for further research.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003eThis is a retrospective cohort study that examined the medical records of women with diminished ovarian reserve (DOR) or poor ovarian response (POR) who underwent controlled ovarian stimulation (COS) and embryo transfer (ET) using vitrified embryos. The study occurred at our institution from June 1, 2019, to December 31, 2022.\u003c/p\u003e \u003cp\u003eDOR was defined as having a low antral follicle count (AFC\u0026thinsp;\u0026lt;\u0026thinsp;5) or a low serum anti-M\u0026uuml;llerian hormone (AMH) level (\u0026lt;\u0026thinsp;1.2 ng/ml) at the start of ovarian stimulation. The study included women in Poseidon Groups 3 and 4 with at least one vitrified zygote with two pronuclei (2PN) created for intended transfer after multiple stimulation cycles. Exclusion criteria were the use of donor oocytes, preimplantation genetic testing (PGT), and failure to retrieve oocytes after COS. All intracytoplasmic sperm injection (ICSI) was performed due to male factors.\u003c/p\u003e \u003cp\u003eThe study evaluated patient characteristics such as age, body mass index, family history, primary or secondary infertility, cause of infertility, and AMH levels. The decision to stop zygote accumulation was made through a discussion between the patient and the physician. The Institutional Review Board and the Chang Gung Memorial Hospital Ethics Committee in Kaohsiung, Taiwan, approved the study (IRB No.: 202400767B0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eProtocols\u003c/h2\u003e \u003cp\u003eThe ovarian stimulation protocols included gonadotropin-releasing hormone (GnRH) antagonist protocols and progestin-primed ovarian stimulation protocols, all performed following standard clinical practice. Each protocol was selected based on factors such as ovarian reserve, age, and previous ovarian response. The dose of gonadotropin was adjusted based on the individual response. After the maturation of follicles, oocytes were retrieved, and standard IVF or ICSI procedures were used for fertilization, as previously described[25, 26].\u003c/p\u003e \u003cp\u003eThe presence of clear 2PNs was confirmed prior to the start of vitrification to avoid vitrifying zygotes in syngamy. The details of oocyte grading assessment of fertilization, embryo culture, zygote, embryo grading, endometrial preparation for frozen embryo transfer are described elsewhere [27].\u003c/p\u003e \u003cp\u003eThe choice between Day 3 embryo transfer and extended culture to blastocyst and transfer on Day 5 was based on embryo quality and number[28].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eVitrification\u003c/h2\u003e \u003cp\u003eThe method of zygote vitrification/warming was described by Kuwayama et al. [29]. We used the Cryotech Vitrification/warming kit according to the manufacturer\u0026rsquo;s protocols.\u003c/p\u003e \u003cp\u003eTransfer the Zygote on the surface of ES (Equilibration Solution) for 12-15min; after complete equilibration, transfer the Zygote to the VS (Vitrification Solution) for 60-90sec, then place the Zygote near the black mark on the Cryotec sheet with small volume, immediately submerge and stir the Cryotec in fresh liquid nitrogen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWarming\u003c/h2\u003e \u003cp\u003eQuickly put the Cryotec from liquid nitrogen into the TS for l min, then aspirate the Zygote to the bottom of the DS (Dilution solution) well for 3 min. Wash the Zygote to WS1(Washing Solution ) for 5 min, then WS2 for 1 min. Then, culture the zygote in Universal IVF Medium (ORIGIO) for 1hr then transfer to the sequential medium G1/G 2 (Vitrolife) media until cleavage stage or blastocyst stage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCosts\u003c/h2\u003e \u003cp\u003eThe costs were based on the actual cost for each patient, as recorded in their clinical records. The oocyte pickup (OPU) cost included charges for procedures regarding oocyte retrieval, pre-op tests, anesthesia, insemination, and embryo culture costs. The costs for the hormonal test and sonography included the test on the FSH, estradiol, progesterone, LH, and sonography during the COH cycle. Costs for COH medication included drugs for COH and the triggering of ovulation. Costs of embryo transfer, including vitrification of zygote, embryo thawing, medication for endometrium priming, monitoring with sonography, hormonal tests, embryo transfer, and luteal phase support after ET, all of which had accumulated until the day of the urinary hCG test 2 weeks after embryo transfer. All charges were calculated in Taiwanese Dollars (NTD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOutcome measures\u003c/h2\u003e \u003cp\u003eThe study focused on outcome measures, with the primary outcome being clinical pregnancy, defined as the presence of gestational sac(s) on transvaginal ultrasound after 6\u0026ndash;7 weeks of gestation. Clinical abortion was recorded in cases of pregnancy loss between the clinical detection of pregnancy and the 22nd week of gestation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as mean and standard deviation and compared using Mann\u0026ndash;Whitney U tests. Categorical variables were expressed as proportion and percentages and compared using Fisher's exact test. The effects of potential clinical and laboratory factors were examined, and the statistical significance of each variable was evaluated using univariate analysis. A \u003cem\u003ep\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant in all analyses\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eWe have reviewed the medical records of 278 IVF/ICSI cycles conducted on 122 women at our institution over a 2-year period. Table\u0026nbsp;1 shows the characteristics of these patients and the types of assisted reproduction techniques used.\u003c/p\u003e \u003cp\u003eThe average age of the patients was 40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 years, and the mean serum AMH level was 0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 ng/mL. Of the cycles, 22 (7.9%) resulted in no oocytes being retrieved, while 256 (92.1%) had at least one oocyte retrieved for culture. Only one woman (0.8%) did not have an embryo for transfer because she stopped the treatment protocol after a failed oocyte retrieval stimulation. The clinical pregnancy rate was 22.3% per transfer and 11.2% per cycle (Table\u0026nbsp;2). We divided all cycles into two groups based on maternal age using the Poseidon classification: those where the female was \u0026lt;\u0026thinsp;40 years (n\u0026thinsp;=\u0026thinsp;51) and those where the female was \u0026ge;\u0026thinsp;40 years (n\u0026thinsp;=\u0026thinsp;71) (Table\u0026nbsp;3). Our results demonstrated that the younger group had significantly more favorable outcomes compared to the older group, particularly in live birth rates per cycle (12.6% vs. 4%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029) and per transfer (24.1% vs. 9.9%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027).\u003c/p\u003e \u003cp\u003eHowever, the older group had more embryos per transfer (2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 vs. 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014). There were no differences between the number of stimulation cycles per patient, the number of oocytes and mature oocytes retrieved, the number of transfer cycles per patient, the cost per patient, or per transfer. The younger group seemed to have a better clinical pregnancy rate per cycle (15.5% vs. 8.4%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.066) and per transfer (29.6% vs. 21.1%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.088), but both showed no significance.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;4 shows the costs of zygote accumulations. We set a model mimicking the cost of fresh embryo transfer with the same number of stimulation and embryo transfer cycles. The costs of the zygote accumulation strategy were higher both per patient (NTD 257,164 vs. 161,508) and per cycle (NTD 225,066 vs. 141,755) than those of the fresh embryo transfer model.\u003c/p\u003e \u003cp\u003eFigure 1 shows the difference (maximum minus the minimum number) of oocyte retrieval and pronuclei (2PN) formation in the same patient treated with different ovarian stimulation. 82.8% (101/122) and 90%(112/125) of patients\u0026rsquo; differences in oocyte retrieval and pronuclei (2PN) formation varied between 0 and 2.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis retrospective study, we found that younger women undergoing vitrified zygote accumulation strategy with diminished ovarian reserve had significantly better live birth rates than the older women, even though the older group had a higher number of embryos per transfer. The zygote accumulation strategy might cost more than fresh embryo transfer if the number of stimulations and embryo transfer cycles are the same in a patient. The main difference in the cost includes vitrification of the zygote, embryo thawing, medication for endometrium preparation, monitoring with sonography, and hormonal tests.\u003c/p\u003e \u003cp\u003ePatients included in our study were poor responders (median AMH\u0026thinsp;=\u0026thinsp;0.78 ng/mL) with advanced age (median age\u0026thinsp;=\u0026thinsp;40), and these patients are challenging for physicians to deal with. Our study is one of the few to deal with such low ovarian reserve and older women undergoing repeated zygote freezing with autologous oocytes from IVF/ICSI. Besides, this is the first study to report the outcome and costs of the zygote accumulation strategy. The outcome and the price may represent the effectiveness of zygote accumulation in women with extremely diminished ovarian reserve.\u003c/p\u003e \u003cp\u003eSu et al. [27],, in a previous study in our institution, investigated females from whom zero, one, two, or three oocytes were retrieved following fresh IVF/ICSI. All cycles were divided according to maternal age using the Bologna criteria into those in which the female was \u0026lt;\u0026thinsp;40 years (n\u0026thinsp;=\u0026thinsp;592) and \u0026ge;\u0026thinsp;40 years (n\u0026thinsp;=\u0026thinsp;266). The results demonstrated that the younger group had significantly more favorable clinical pregnancy rates than the older group. Our study also divided women into \u0026lt;\u0026thinsp;40 and \u0026ge;\u0026thinsp;40 years old (Table\u0026nbsp;5), and we had a similar average age in both groups. Besides, in the younger group (\u0026lt;\u0026thinsp;40 years old) compared to a previous study, we reported lower clinical pregnancy rate per cycle (15.5%[16/103] vs. 20.7%[123/592]) and live birth rate per cycle (12.6%[13/103] vs. 17.2%[102/592]), but better clinical pregnancy rate per transfer (29.6%[16/54] vs. 27.7%[123/444]) and live birth rate per transfer (24.1%[13/54] vs. 23%[102/444]). On the other hand, our study demonstrated better outcomes in the advanced age group with clinical pregnancy rate per transfer (21.1%[15/71] vs. 7.6%[14/185]), clinical pregnancy rate per cycle (8.4%[15/175] vs. 5.3%[14/266]), live birth rate per transfer (9.9%[7/71] vs. 5.4% [10/185]) and live birth rate per cycle(4% [7/175] vs. 3.8% [10/266]). These results may indicate that zygote accumulation strategy should be considered in older females undergoing IVF/ICSI.\u003c/p\u003e \u003cp\u003eIn a study by Datta et al. [21], the results of transferring frozen embryos collected over three consecutive modified natural cycles in women with low ovarian reserve were examined. It was found that the live birth rate after a single embryo transfer following three consecutive modified natural cycles was significantly higher compared to a fresh embryo transfer in a single modified natural IVF cycle (30.6% vs. 13.3% respectively; p\u0026thinsp;=\u0026thinsp;0.002). Additionally, the cumulative live birth rate for all embryo transfers was also notably higher in the accumulation group. However, it's important to note that there may be some bias in this comparison, as it was based on all transfers from embryo accumulation over three stimulation cycles versus a fresh embryo transfer after one stimulation cycle.\u003c/p\u003e \u003cp\u003eOn the other hand, based on the Bologna criteria, Ge et al. implemented a different approach to collecting embryos from women with a poor response undergoing IVF/ICSI treatment. In their study, all couples were given multiple stimulation cycles until they had at least five embryos for transfer. The findings revealed that embryo banking in women with poor ovarian response led to a significantly lower cumulative live birth rate and a longer time to achieve live birth compared to not using embryo banking. The conclusion drawn was that employing an embryo banking strategy for poor responders is less effective than a conventional non-banking approach[22].\u003c/p\u003e \u003cp\u003eOur study's selection of the POSEIDON criteria differed from those used in the previously mentioned two studies, Datta et al. [21] and Ge et al. [22], which adopted the Bologna criteria. Moreover, the methods of embryo accumulation were different compared with the two studies. Datta et al. transferred accumulated frozen embryos after three consecutive modified natural cycles in women with low ovarian reserve, regardless of the number of embryos obtained. On the other hand, Ge et al. provided several stimulation cycles until at least five embryos were available for transfer, so every participant had a chance for embryo transfer. The average stimulation cycle per woman was 5.3 to accumulate five embryos available for transfer. In comparison, we vitrified the zygotes rather than the embryos. In addition, we didn't set a goal for the number of stimulation cycles or the number of embryos available for transfer. We let the patient and the physician decide when to stop the zygote accumulation program according to the physician's expertise and the patient's financial, physical, and mental conditions. It was more representative to reflect routine clinical practice in which poor responders may be present.\u003c/p\u003e \u003cp\u003eIt is reasonable that the dropout rate and the number of patients without embryos for transfer were low when the embryo and zygote accumulation strategy were applied. In the Datta et al. study, nine patients (7.2%) had no embryos for transfer with zero dropout incidence[21]. On the other hand, every patient in the banking group completed the study and had a transferable embryo, as shown in Ge et al.\u0026rsquo;s report[22]. In our study, only one woman (0.8%) had no embryo for transfer and dropped out, with the average stimulation cycles per woman being 2.3.\u003c/p\u003e \u003cp\u003eInfertile couples experience pressure from society, family, and treatment and have a higher risk of negative emotions, such as anxiety and depression [30\u0026ndash;32]. Stress and anxiety levels remained elevated across all cycles. Women with lower stress and anxiety levels on the day before oocyte retrieval had a higher pregnancy rate[33]. Since the average stimulation cycles per patient were 2.3 in our study and the couples don\u0026rsquo;t need to confront the outcome of embryo transfer as much as fresh transfer, we might presume that the stress and anxiety levels were lower with the zygote accumulation strategy. Also, it might contribute to the low dropout rate.\u003c/p\u003e \u003cp\u003eAfter controlled ovarian stimulation, we also reported the variation in oocyte and pronuclei (2PN) yield. More than 80 percent of patients\u0026rsquo; differences (maximum minus the minimum number) in oocyte retrieval and pronuclei (2PN) formation varied between 0 and 2 (Fig.\u0026nbsp;1). This result implies that a patient may fluctuate in follicle recruitment during different menstrual cycles.\u003c/p\u003e \u003cp\u003eResearchers Rustamov et al. discovered that, even with personalized treatment, there is a limit to how much variation in response can be minimized[34]. They found that there could be a 3.4-fold difference in yield between two treatment cycles for patients with similar characteristics. Similarly, when the same patient was treated in the same way on two occasions, a 2.7-fold difference in yield could be observed. These findings have significant clinical implications. For instance, if a patient obtained nine oocytes from a cycle, it can be expected that another patient with similar characteristics and similar treatment would produce between 6 and 13 oocytes. Any response in the range of 4 to 19 oocytes would not be surprising. If the same patient were stimulated in the same way a second time, it could be anticipated that the number of oocytes retrieved would fall between 7 and 12, with an expected range of 5 to 17 oocytes.\u003c/p\u003e \u003cp\u003eThe limitations of our study are its retrospective design and being performed at a single center, so results may need to be more generalizable to other populations and clinical settings. Also, although we have the same team that has previously published a non-accumulating zygote strategy for similar patients for comparison, we didn\u0026rsquo;t set a well-designed control group so that the outcome couldn\u0026rsquo;t be compared to other methods. Randomized controlled trials with adequate sample size should be conducted to confirm these findings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, by applying the vitrified zygote accumulation strategy in women with a poor response based on the POSEIDON criteria starting their IVF/ICSI treatment, younger women had significantly better live birth rates than the older women, even though the older group had a higher number of embryos per transfer. However, the zygote accumulation strategy benefits older women more than fresh embryo transfer. The strategy might cost more but decrease the anxiety and stress.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e2PN: two pronuclei; AFC :Antral follicle count; AMH :Anti‑M\u0026uuml;llerian hormone; COS :Controlled ovarian stimulation; DOR: Diminished ovarian reserve; ET: Embryo transfer; ICSI:Intracytoplasmic sperm injection; IVF: In vitro fertilization; GnRH: Gonadotropin‑releasing hormone; PGT: Preimplantation genetic testing; POR :Poor ovarian reserve\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the staff of Kaohsiung Chang Gung Memorial Hospital for the tribute and the involvement in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eK‑C L, and P‑T H contributed to the conception and design of the study, collecting patients in Kaohsiung Chang Gung Memorial Hospital and data analysis as well as interpretation. P-T H, N-C T, Y-J L, H-T L, Y-Y H, Y-T S, H-W H, K-C L were involved in discussion and advising the study design. K‑C L was the co‑corresponding author and played the major role in the design of manuscript, interpreting the analyzed data, advising, drafting, and revising the draft. All authors were involved in the writing of the manuscript and provided final approval.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This study was supported by CMRPG8M0241-2 from Chang Gung Memorial Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Institutional Review Board and the Chang Gung Memorial Hospital Ethics Committee in Kaohsiung, Taiwan, approved the study (IRB No.: 202400767B0). There is no participants\u0026rsquo; consent for this retrospective study and Institutional Review Board decides this is ethically acceptable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFerraretti, A.P., et al., \u003cem\u003eESHRE consensus on the definition of 'poor response' to ovarian stimulation for in vitro fertilization: the Bologna criteria.\u003c/em\u003e 2011(1460-2350 (Electronic)).\u003c/li\u003e\n\u003cli\u003ePoseidon, G., et al., \u003cem\u003eA new more detailed stratification of low responders to ovarian stimulation: from a poor ovarian response to a low prognosis concept.\u003c/em\u003e Fertil Steril, 2016. \u003cstrong\u003e105\u003c/strong\u003e(6): p. 1452-3.\u003c/li\u003e\n\u003cli\u003eVerberg, M.F., et al., \u003cem\u003eWhy do couples drop-out from IVF treatment? A prospective cohort study.\u003c/em\u003e 2008(1460-2350 (Electronic)).\u003c/li\u003e\n\u003cli\u003eDrakopoulos, P., et al., \u003cem\u003eConventional ovarian stimulation and single embryo transfer for IVF/ICSI. How many oocytes do we need to maximize cumulative live birth rates after utilization of all fresh and frozen embryos?\u003c/em\u003e 2016(1460-2350 (Electronic)).\u003c/li\u003e\n\u003cli\u003eSunkara, S.K., et al., \u003cem\u003eAssociation between the number of eggs and live birth in IVF treatment: an analysis of 400 135 treatment cycles.\u003c/em\u003e 2011(1460-2350 (Electronic)).\u003c/li\u003e\n\u003cli\u003evan der Gaast, M.H., et al., \u003cem\u003eOptimum number of oocytes for a successful first IVF treatment cycle.\u003c/em\u003e Reprod Biomed Online, 2006(1472-6483 (Print)).\u003c/li\u003e\n\u003cli\u003eGiovanale, V., et al., \u003cem\u003ePoor responders in IVF: an update in therapy.\u003c/em\u003e Gynecological Endocrinology, 2015(1473-0766 (Electronic)).\u003c/li\u003e\n\u003cli\u003eHaahr, T., et al., \u003cem\u003eManagement Strategies for POSEIDON Groups 3 and 4.\u003c/em\u003e Front Endocrinol (Lausanne), 2019. \u003cstrong\u003e10\u003c/strong\u003e: p. 614.\u003c/li\u003e\n\u003cli\u003eJin, B., et al., \u003cem\u003eComparison of clinical outcomes among dual ovarian stimulation, mild stimulation and luteal phase stimulation protocols in women with poor ovarian response.\u003c/em\u003e Gynecological Endocrinology, 2018(1473-0766 (Electronic)).\u003c/li\u003e\n\u003cli\u003eVaiarelli, A., et al., \u003cem\u003eWhat is new in the management of poor ovarian response in IVF?\u003c/em\u003e Current opinion in obstetrics \u0026amp; gynecology, 2018(1473-656X (Electronic)).\u003c/li\u003e\n\u003cli\u003eCobo, A., et al., \u003cem\u003eComparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method.\u003c/em\u003e Fertility and sterility, 2008(1556-5653 (Electronic)).\u003c/li\u003e\n\u003cli\u003eCobo, A., et al., \u003cem\u003eUse of cryo-banked oocytes in an ovum donation programme: a prospective, randomized, controlled, clinical trial.\u003c/em\u003e Human reproduction, 2010(1460-2350 (Electronic)).\u003c/li\u003e\n\u003cli\u003eParmegiani, L., et al., \u003cem\u003eEfficiency of aseptic open vitrification and hermetical cryostorage of human oocytes.\u003c/em\u003e Reproductive biomedicine online, 2011(1472-6491 (Electronic)).\u003c/li\u003e\n\u003cli\u003eRienzi, L., et al., \u003cem\u003eEmbryo development of fresh 'versus' vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study.\u003c/em\u003e Human reproduction, 2010(1460-2350 (Electronic)).\u003c/li\u003e\n\u003cli\u003eAlmodin, C.G., et al., \u003cem\u003eEmbryo development and gestation using fresh and vitrified oocytes.\u003c/em\u003e Human reproduction, 2010(1460-2350 (Electronic)).\u003c/li\u003e\n\u003cli\u003eDomingues, T.S., et al., \u003cem\u003eEgg donation of vitrified oocytes bank produces similar pregnancy rates by blastocyst transfer when compared to fresh cycle.\u003c/em\u003e Journal of assisted reproduction and genetics, 2017(1573-7330 (Electronic)).\u003c/li\u003e\n\u003cli\u003eDoyle, J.O., et al., \u003cem\u003eSuccessful elective and medically indicated oocyte vitrification and warming for autologous in vitro fertilization, with predicted birth probabilities for fertility preservation according to number of cryopreserved oocytes and age at retrieval.\u003c/em\u003e Fertility and sterility, 2016(1556-5653 (Electronic)).\u003c/li\u003e\n\u003cli\u003eRienzi, L., et al., \u003cem\u003eOocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance.\u003c/em\u003e 2017(1460-2369 (Electronic)).\u003c/li\u003e\n\u003cli\u003eCobo, A., et al., \u003cem\u003eAccumulation of oocytes: a new strategy for managing low-responder patients.\u003c/em\u003e Reprod Biomed Online, 2012. \u003cstrong\u003e24\u003c/strong\u003e(4): p. 424-32.\u003c/li\u003e\n\u003cli\u003eLee, K.S., et al., \u003cem\u003eThe live birth rate of vitrified oocyte accumulation for managing diminished ovarian reserve: a retrospective cohort study.\u003c/em\u003e J Ovarian Res, 2023. \u003cstrong\u003e16\u003c/strong\u003e(1): p. 49.\u003c/li\u003e\n\u003cli\u003eDatta, A.K., et al., \u003cem\u003eAccumulation of embryos over 3 natural modified IVF (ICSI) cycles followed by transfer to improve the outcome of poor responders.\u003c/em\u003e Facts Views Vis Obgyn, 2019. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 77-84.\u003c/li\u003e\n\u003cli\u003eGe, Q.L., et al., \u003cem\u003eThe impact of the embryo banking on the cumulative live birth rate in women with poor ovarian response according to the Bologna criteria.\u003c/em\u003e Reprod Med Biol, 2023. \u003cstrong\u003e22\u003c/strong\u003e(1): p. e12533.\u003c/li\u003e\n\u003cli\u003eGolakov, M.A.-O., et al., \u003cem\u003eWhat is the net effect of introducing vitrification for cryopreservation of surplus 2PN oocytes in an IVF program?\u003c/em\u003e 2018(1432-0711 (Electronic)).\u003c/li\u003e\n\u003cli\u003eShapiro, B.S., et al., \u003cem\u003eFreeze-all at the blastocyst or bipronuclear stage: a randomized clinical trial.\u003c/em\u003e 2015(1556-5653 (Electronic)).\u003c/li\u003e\n\u003cli\u003eTsai, N.C., et al., \u003cem\u003eMonopronucleated (1PN) and tripronuclear (3PN) zygotes formation during assisted reproduction in POSEIDON group 4 patients: Emphasizing on polar bodies.\u003c/em\u003e 2021(1447-0756 (Electronic)).\u003c/li\u003e\n\u003cli\u003eTsai, N.C., et al., \u003cem\u003eDevelopmental potential of surplus morulas with delayed and/or incomplete compaction after freezing-thawing procedures.\u003c/em\u003e Reprod Biol Endocrinol, 2019. \u003cstrong\u003e17\u003c/strong\u003e(1): p. 87.\u003c/li\u003e\n\u003cli\u003eSu, Y.T., et al., \u003cem\u003eAge is a major prognosticator in extremely low oocyte retrieval cycles.\u003c/em\u003e Taiwan J Obstet Gynecol, 2017. \u003cstrong\u003e56\u003c/strong\u003e(2): p. 175-180.\u003c/li\u003e\n\u003cli\u003eLan, K.C., et al., \u003cem\u003eThe predictive value of using a combined Z-score and day 3 embryo morphology score in the assessment of embryo survival on day 5.\u003c/em\u003e Hum Reprod, 2003. \u003cstrong\u003e18\u003c/strong\u003e(6): p. 1299-306.\u003c/li\u003e\n\u003cli\u003eKuwayama, M., \u003cem\u003eHighly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method.\u003c/em\u003e Theriogenology, 2007. \u003cstrong\u003e67\u003c/strong\u003e(1): p. 73-80.\u003c/li\u003e\n\u003cli\u003eBeukers, F., et al., \u003cem\u003eParental psychological distress and anxiety after a successful IVF/ICSI procedure with and without preimplantation genetic screening: follow-up of a randomised controlled trial.\u003c/em\u003e 2012(1872-6232 (Electronic)).\u003c/li\u003e\n\u003cli\u003eLoGiudice, J.A. and J. Massaro, \u003cem\u003eThe impact of complementary therapies on psychosocial factors in women undergoing in vitro fertilization (IVF): A systematic literature review.\u003c/em\u003e 2017(1532-8201 (Electronic)).\u003c/li\u003e\n\u003cli\u003eHaimovici, F.A.-O., et al., \u003cem\u003eStress, anxiety, and depression of both partners in infertile couples are associated with cytokine levels and adverse IVF outcome.\u003c/em\u003e 2018(1600-0897 (Electronic)).\u003c/li\u003e\n\u003cli\u003eTurner, K., et al., \u003cem\u003eStress and anxiety scores in first and repeat IVF cycles: a pilot study.\u003c/em\u003e PLoS One, 2013. \u003cstrong\u003e8\u003c/strong\u003e(5): p. e63743.\u003c/li\u003e\n\u003cli\u003eRustamov, O., et al., \u003cem\u003eHow much variation in oocyte yield after controlled ovarian stimulation can be explained? A multilevel modelling study.\u003c/em\u003e 2017(2399-3529 (Electronic)).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1-5 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diminished ovarian reserve (DOR), Embryo transfer, Vitrification, Zygote accumulation","lastPublishedDoi":"10.21203/rs.3.rs-4573045/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4573045/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAccumulated vitrified embryos are stored for later thawed transfer to deal with poor responders. However, the cost and outcome of vitrified zygotes have yet to be studied. Our study aimed to investigate the effectiveness of the vitrified zygote accumulation method in diminished ovarian reserve patients.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis retrospective study included 122 diminished ovarian reserve patients who met the Poseidon classification groups 3 and 4 from June 1, 2019, to December 31, 2022. The patients underwent controlled ovarian stimulation and embryo transfer using accumulated vitrified zygotes. The clinical pregnancy rate and live birth rate per embryo transfer and stimulation cycle are evaluated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTwo hundred seventy-eight IVF/ICSI cycles in one hundred twenty-two females treated at our institution were divided into two groups, according to age\u0026thinsp;\u0026lt;\u0026thinsp;40 years (n\u0026thinsp;=\u0026thinsp;51) and \u0026ge;\u0026thinsp;40 years (n\u0026thinsp;=\u0026thinsp;71). The younger group had significantly more favorable outcomes than the older group only in live birth rates per cycle (12.6% vs. 4%, p\u0026thinsp;=\u0026thinsp;0.029) and per transfer (24.1% vs. 9.9%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027). However, the older group had more embryos per transfer (2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 vs. 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014). There were no differences between the number of stimulation cycles per patient, the number of oocytes and mature oocytes retrieved, the number of transfer cycles per patient, the cost per patient, or transfer. We set a model mimicking the cost of fresh embryos. The zygote accumulation strategy costs were higher per patient and per cycle than those of the fresh embryo transfer model.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn diminished ovarian reserve women undergoing IVF/ICSI treatment, younger women had significantly better live birth rates than the older women applying the vitrified zygote accumulation strategy, even though the older group had a higher number of embryos per transfer. However, the zygote accumulation strategy benefits older women more than fresh embryo transfer. The strategy might cost more but decrease the anxiety and stress.\u003c/p\u003e","manuscriptTitle":"The effectiveness of vitrified zygote accumulation for managing the extremely diminished ovarian reserve in infertile women: a retrospective cohort study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-13 01:47:33","doi":"10.21203/rs.3.rs-4573045/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"10207347-353e-49f7-a750-ce896c27c5cb","owner":[],"postedDate":"July 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-16T15:53:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-13 01:47:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4573045","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4573045","identity":"rs-4573045","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}