Minimal Stimulation and Natural Cycle In Vitro Fertilization, 1st ed. 2015

11. Vitrification Is the Keystone to Minimal Stimulation IVF

Goral Gandhi  and Gautam N. Allahbadia 


Department of Assisted Reproduction, Rotunda – The Center for Human Reproduction, 36, Turner Road, B-Wing, 101, Bandra W, Mumbai, Maharashtra, 400 050, India


Department of Assisted Reproduction, Rotunda-Blue Fertility Clinic & Keyhole Surgery Center, Rotunda – The Center for Human Reproduction, 36, Turner Road, B-Wing, 101, Bandra W, Mumbai, Maharashtra, 400 050, India

Goral Gandhi (Corresponding author)



Gautam N. Allahbadia




Minimal stimulation in vitro fertilization (IVF) has several benefits over conventional IVF protocols, including less medication, fewer injections, and producing fewer but better quality eggs. The use of Clomiphene citrate in minimal stimulation IVF protocols has a negative impact on the endometrium. Cryopreserved embryo transfers with this protocol have yielded much higher pregnancy rates compared to fresh transfers. Minimal stimulation cycle with a remote embryo transfer (rET) is a viable option only when it is combined with an efficient embryo cryopreservation program. The recent use of the vitrification technique has shown a higher embryo survival rate, compared with slow freezing, resulting in significantly higher implantation and pregnancy rates per transfer. Therefore, the use of elective cryopreservation of viable embryos could be an alternative to avoid the deleterious effects of the stimulation cycles on embryo–endometrium synchrony.


Minimal stimulationVitrificationRemote cycle embryo transferEmbryo cryopreservationClomiphene citrateMini IVFIVF LiteACCU-VITLow responders


Embryo implantation is one of the important steps for the success of assisted reproduction techniques (ART) (Díaz-Gimeno et al. 2011). Its effectiveness relies on three main parameters: embryo quality, endometrial receptivity (ER), and a well-balanced embryo–endometrium interaction (Achache and Revel 2006). The implantation window is a self-limited period in which the endometrium acquires adequate morphologic and functional state for the blastocyst attachment. Implantation failure remains an unsolved problem in ART. In two-thirds of the implantation failures, the primary cause of failure is the impairment of the ER, whereas the embryo itself is responsible for only one-third of the failures (Achache and Revel 2006).

Therefore, ER is essential for conception in natural and infertility treatment cycles. However, it has been suggested that controlled ovarian hyperstimulation (COH) adversely affects ER during ART cycles (Bourgain and Devroey 2003; Devroey et al. 2004; Kolibianakis et al. 2002). In fresh cycles with COH, the elevated progesterone (P) levels cause morphologic and biochemical endometrial alterations leading to advanced endometrium maturation compared with the natural cycle (Shapiro et al. 2010a; Al-Azemi et al. 2012). There is also evidences showing that high E2 levels (>2500 pg/mL) may impair the endometrial maturation and implantation (Simon et al. 1995; Groothuis et al. 2007). These altered hormone levels could mediate an asynchrony between the endometrium and the transferred embryos, leading to implantation failure (Nikas et al. 1999; Simon et al. 1998).

In ART, the highest pregnancy rates are obtained in fresh oocyte donation cycles. In these cycles, the endometrium is artificially primed and the embryos are therefore, transferred to an environment that had not suffered the effects of the supraphysiologic hormonal levels that occur during COH (Shapiro et al. 2009). Although the oocytes are of the same quality, some studies of shared oocyte cycles found significantly higher pregnancy rates in recipients compared with oocyte donors, and this may be related to a superior quality of ER (Simon et al. 1998; Shapiro et al. 2009).

The use of frozen embryo transfer (FET), compared with fresh embryo transfer significantly improves clinical and ongoing pregnancy rates (Roque et al. 2013). Endometrial preparation is better achieved during natural cycles or with hormone replacement therapy with exogenous estradiol (E2) and P, compared with stimulated cycles (Shapiro et al. 2009; Paulson et al. 1990; Paulson 2011). It is suggested that during the endometrial priming for FET, the endometrium is more receptive than in fresh embryo cycles (Mandelbaum 2000; Martínez-Conejero et al. 2007). With advances in embryo cryopreservation techniques, the quality of the frozen embryos and their potential for implantation are similar to that observed with fresh embryos (Shapiro et al. 2010b2011). The cryopreservation of all embryos has become a routine procedure in ART when embryo transfer is either impossible or inconvenient.

Minimal Stimulation and Endometrium Receptivity

The aim of any medical treatment is to provide its patients with a safe and cost-effective treatment, which results in a positive outcome. Therefore, gentle stimulation protocols, such as minimal stimulation IVF, can be a good option. The minimal stimulation protocols were evolved with the intention of providing a more natural stimulation for IVF. Minimal stimulation protocols have the potential of reducing the complications as well as the overall cost of conventional IVF (Philips et al. 2000). Minimal stimulation protocols have shown to have many benefits over conventional IVF protocols, the main one being production of fewer but better quality oocytes (Devreker et al. 1999; Fauser et al. 1999). Minimal stimulation can be beneficial for management of patients exhibiting poor or hyperovarian response. However, it can be offered to all groups of patients, including young patients with a good prognosis, poor responders, and women of advanced age, as an alternative to conventional protocols.

In a minimal stimulation protocol, Clomiphene citrate, an antiestrogen, is used to increase the number of developed follicles by elevating endogenous FSH. Clomiphene citrate has been traditionally used as the drug of choice in treating women with anovulatory infertility (Greenblatt et al. 1961). Clomiphene citrate contains an unequal mixture of two isomers, Enclomiphene and Zuclomiphene, as citrate salts. Zuclomiphene is much more potent for inducing ovulation. Clomiphene citrate induces follicle-stimulating hormone (FSH) release from the anterior pituitary, and this is often enough to reset the cycle of events leading to ovulation. Continuation of Clomiphene citrate for more than 5 days was also able to inhibit the LH surge (Teramoto and Kato 2007). However, Clomiphene citrate treatment has shown to have adverse effects on the endometrium receptivity (Dehbashi et al. 2003) and increased incidence of luteal phase deficiency (Fritz et al. 1991). This is due to the antiestrogenic effect of Clomiphene. Clomiphene induces a decrease in endometrial glandular size and number in both anovulatory and in normo-ovulatory women, and the effect remains even after human chorionic gonadotropin (hCG) administration to induce final oocyte maturation. This results in lower per cycle pregnancy rate compared to conventional protocols.

Proponents of the minimal stimulation method cite a cumulative success rate similar to a single cycle of conventional IVF (Moragianni and Penzias 2010). As compared to the conventional IVF protocol, the minimal stimulation method has increased patient tolerance and acceptability to multiple cycles. Improved pregnancy rates using minimal stimulation IVF can be achieved with frozen embryo transfer as compared to fresh transfers (Zhang et al. 2010). Use of Clomiphene citrate, with its negative impact on the endometrium, requires a reliable method of embryo cryopreservation. With minimal stimulation yielding few embryos per cycle, it has to be ensured that these embryos survive the cryopreservation process. A highly efficient vitrification program is extremely critical to minimize embryo loss and maximize the chances of pregnancy.


Embryo cryopreservation has been practised routinely and forms an integral part of IVF treatment. However, it is important to acknowledge that the term “cryopreservation” does not imply a universal freezing technique, it may be divided into two large groups: slow rate freezing and vitrification. Several factors, such as exposure time of cells to the different cryoprotectant solutions and their concentrations, as well as the rate of formation of extra- and intracellular ice crystals, significantly affect the survival rate of the post-warmed embryos. Therefore, selection of a suitable technique plays a key role in the success of the cryopreservation procedure.

Slow rate freezing has been routinely used to cryopreserve gametes and embryos for many years. During cryopreservation, temperature is gradually dropped from +37 to −196 °C, to create preferential crystal formation outside the cell. This method, however, poses a drawback of dehydration and ice crystal formation. The concentration of solutes increases considerably, triggering the possibility of osmotic shock. Ice crystal formation, as well as the chilling effects seen with slow cooling, can damage cells and may exacerbate the toxicity of cryoprotectants, leading to fracture damage and other lethal injuries (Rall and Fahy 1985). Furthermore, if cells survive freezing, they might sustain additional damage during the thawing process due to osmotic imbalance, uncontrollable swelling, and ice recrystallization (Woods et al. 2004). Hence, the use of the slow freezing method has become controversial due to its difficulties, expense, and respective low success rates in ART (Kuleshova and Lopata 2002).

Vitrification was developed to overcome the harmful effects of ice crystal formation that occurs during the slow freezing method. Vitrification can be defined as a physical process by which a highly concentrated solution of cryoprotectant solidifies into a glassy vitrified state by an extreme elevation in the viscosity while cooling at a low temperature. The cryoprotectant, in this state of high viscosity, continues to retain its normal molecular and ionic distribution of liquid state and can be considered to be a supercooled liquid (Luyet 1937a). The process negates the formation of intracellular and extracellular ice crystals (Luyet 1937b). It thus alleviates the potential damage that can be caused by intracellular ice formation and the osmotic effects related to extracellular ice formation. Extremely high cooling rates are achieved by direct plunging into liquid nitrogen with a minimal volume (≤0.5 mL) of final vitrification solution including vitrified cells. Such high cooling results in a high survival rate and better viability and helps escape ice crystal formation even with the lower concentration of cryoprotectant agent (CPA).

The vitrification strategy in itself can vary greatly, with differing protocols relating to cryoprotectants, warming and cooling procedures. There are also two distinct systems for vitrification: closed and open systems. High cooling rates with vitrification can be achieved through the use of carriers that allow cryopreservation in fluid volumes <1 μl. Open carriers allow direct contact of embryos with liquid nitrogen (LN2), whereas closed carrier systems sequester the embryo within a sealed system during immersion in LN2. Open system helps achieve high cooling rates compared to closed system. The use of closed systems may be preferable to avoid theoretical concerns regarding cross-contamination during direct exposure and storage of cryopreserved samples in LN2. However, no infections have been demonstrated with oocyte/embryo storage to date.

Cryotech Method of Vitrification

Dr. Masashige Kuwayama has a long history of research in vitrification methods. The minimal volume vitrification method introduced by him is now used in increasing number of laboratories worldwide for oocytes, embryos, and blastocyst vitrification. His latest method, the Cryotech method (Cryotech, Japan), has numerous advantages over his previous methods of vitrification (Stehlik et al. 2005). The carrier device is a filmstrip attached to a plastic handle, also equipped with a cap to cover the filmstrip for safe handling and storage. The vitrification solutions have no added serum or synthetic serum supplements. Hence, there is no risk of serum-derived virus contamination. It is a completely chemically defined solution and is stable for a year at 4–8 °C. It contains trehalose instead of sucrose. This overcomes the problem of endotoxicity due to sucrose (Koliblianakis et al. 2009). The Cryotech vitriplates have a special holder for the cryotec; thus, the focus remains the same while washing the embryos in vitrification solution and placing them on the cryotec (Rezazadeh et al. 2009). All these advantages cumulatively add up to an extremely high embryo survival rate. The carrier device (cryotec) can be used as both open and closed vitrification system (Fig. 11.1).


Fig. 11.1

(a) Cryotech carrier for closed cooling system. (b) Cryotech carrier for open cooling system

Method of vitrification: Oocytes/embryos are placed in equilibration solution and incubated until they have completely recovered from the osmotic shock. Oocytes/embryos are then placed in subsequent vitrification solution, mixed well, and after 60s, loaded on the film strip. Then the film part should be submerged into liquid nitrogen with quick and continuous vertical movement to ensure the maximum cooling rate (23,000 °C/min). Finally, under the liquid nitrogen, the cap should be fixed with forceps to protect the film part from mechanical damage during storage.

Method of warming: For warming, the protective cover is removed and the film strip is quickly dipped into the 37 °C warming solution to achieve extremely high warming rate (42,000 °C/min). After 1 min, the solution should be continued in dilution and washing solutions for 3 and 5 min, respectively. The oocytes/embryos are then incubated in culture media.

Vitrification in contrast to slow freezing is an efficient method for cryopreservation as it provides higher survival rate and minimal deleterious effects on post-warming embryo morphology, and it can improve clinical outcomes (Rezazadeh et al. 2009). Vitrification being a more secure method of cryopreservation increases the possibility of using an “all-embryo freeze” or a “single-embryo transfer” protocol, because there is no fear that results with fresh embryos will be better than with cryopreserved (Al-Hasani et al. 2007).

With improvements in cryopreservation technologies, newer applications have emerged making IVF treatments more successful and flexible.

Role of Vitrification in Minimal Stimulation IVF

Zhang et al (2010) described a minimal stimulation protocol christened “mini IVF” which was developed for patients desiring a less stressful and less expensive mode of IVF treatment to achieve a pregnancy. This protocol requires a reliable method for embryo cryopreservation such as vitrification, because of the negative impact of Clomiphene citrate on the endometrium.

The retrospective study included women who underwent a mini-IVF protocol from 2006 to 2009 in New Hope Fertility Center, NY, USA. In this series, patients were not denied treatment based on their day-3 FSH value or ovarian reserve. For fresh embryo transfer cycles, a day-2 or day-3 embryo was transferred, while in cryopreserved transfer cycles, a day-5 or day-6 blastocyst was transferred in a natural cycle or a hormone replacement cycle. The clinical pregnancy rate was better with vitrified–warmed embryo transfer cycles than with fresh embryo transfer cycles (41 % versus 20 %, respectively; P < 0.05). For women under 35 years of age and FSH 15 IU/L for fresh embryo transfers, the pregnancy rate per embryo transfer was 26.8 %, and for cryopreserved embryos, the pregnancy rate per embryo transfer was 47.7 % (P < 0.05). Interestingly, even for FSH > 15 IU/l, the pregnancy rate in women under 35 years of age was 52.6 %, and for those with FSH > 15 IU/l and over 40 years of age, the pregnancy rate per cryopreserved blastocyst transfer was encouragingly as high as 30.8 %. These results strengthen the argument for gentle stimulation protocols and vitrification in preference to standard conventional IVF stimulation protocols.

In a more recent study, Gandhi et al (2013) used an IVF Lite protocol for the treatment of poor ovarian responders. Poor ovarian response (POR) is not a rare occurrence in ovarian stimulation. The incidence of POR is 9.24 % in patients undergoing IVF treatment (Keay et al. 1997). Previous trials have shown that neither conventional IVF nor natural cycle IVF is an effective treatment option for poor ovarian responders (PORs) (Hanoch et al. 1998). Women with poor ovarian reserves, who commonly do not respond to conventional stimulation protocols, are left with few options when planning a family. Tarlatzis et al (2003), in their elegant systematic review, evaluating all the existing ovarian stimulation protocols applied to poor responders, have concluded that the exhausted ovarian apparatus is unable to react to any stimulation, no matter how powerful this might be. The low number of embryos available for transfer poses a great challenge in the management of PORs. A potential management of poor responders is to create a sufficient pool of embryos by accumulating vitrified good-grade embryos over several minimal stimulation cycles (ACCU-VIT). The option of accumulating embryos has become a promising reality with the advent of outstanding vitrification technologies. The study was undertaken to evaluate the efficacy of serial minimal stimulation IVF cycles with vitrification and accumulation of embryos followed by a remote frozen embryo transfer for the treatment of poor ovarian responders as compared to conventional IVF protocols. This approach allows the PORs to have consecutive cycles of embryo accumulation before the follicular reserve is depleted.

The retrospective data analysis included poor ovarian responders from June 2010 and November 2012. A total of 97 PORs underwent treatment with IVF Lite protocol, and 125 PORs underwent treatment with conventional IVF stimulation protocol. The patients identified as PORs based on the Bologna criteria were included in the analysis (Ferraretti et al. 2011). Embryos were vitrified using Cryotech vitrification protocol on day 3. Once six embryos were banked, a frozen embryo transfer was planned and a maximum of three embryos were transferred. The conventional IVF group showed a high ET cancelation rate. Interestingly, there was no significant difference in the number of MII oocytes between the two groups. The IVF Lite group had MII oocytes comparable to the conventional IVF group, with significantly less gonadotropins used. The IVF Lite group had a significantly higher percentage of good-grade embryos than the conventional IVF group. This suggests that when minimal stimulation is used, a cohort of few but better quality oocytes is obtained. The clinical pregnancy rate per embryo transfer was higher in the IVF Lite group (27.81 %) than the conventional IVF group (15.15 %). The cumulative pregnancy rate (CPR) per patient was much higher in the IVF Lite (48.45 %) than the conventional IVF group (24.0 %). The results demonstrate that the IVF Lite protocol consisting of MS-IVF, ACCU-VIT, and rET is a very successful approach in treating poor responders (Tables 11.1 and 11.2).

Table 11.1

Summary of total stimulation cycles


IVF Lite

Conventional IVF

P value

Patients (n)




No. of initiated cycles




Avg no. of initiated cycles/patient




Dosage of gonadotropins (IU)

1646.59 ± 950.78

11349.13 ± 4638.86


No. of retrieval cycles




% canceled retrieval cycles/initiated cycle

14.29 (41/287)

20.22 (56/277)


% cycle with no oocytes retrieved/retrieval cycle

7.32 (18/246)

8.14 (18/221)


% cycle with no fertilization/retrieval cycle

1.63 (4/246)

2.26 (5/221)


Dosage of gonadotropins required/MII oocyte

680.4 (1646.59/2.42)

4956.15 (11349.59/2.29)


Reprinted from Gandhi et al. (2014)

IVF in vitro fertilization, MII metaphase II, Avg average, NS not significant

Table 11.2

Cycle outcomes


IVF lite

Conventional IVF

P value

Patients (n)




No. of transfer cycles (n)




Total embryos/transfer

1.75 ± 0.37

1.77 ± 0.24


Good-grade embryos/transfer

1.52 ± 0.29

1.04 ± 0.46


Clinical pregnancy rate/ET (%)




Clinical pregnancy rate/patient (%)




% cycles with canceled embryo transfers


10.41 (23/221)


Reprinted from Gandhi et al. (2014)

IVF in vitro fertilization, NS not significant, ET embryo transfer


With the dependence of minimal stimulation cycle on Clomiphene citrate, it may be advantageous to cryopreserve all viable embryos and use them in a subsequent FET. The implantation, clinical, and ongoing pregnancy rates of ART cycles may be improved by performing FET compared with fresh embryo transfer. These results may be explained by improved embryo–endometrium synchrony achieved with endometrium preparation cycles, as well as the improved cryopreservation methods (Devroey et al. 2004; Paulson 2011; Mandelbaum 2000). With recent advances, vitrification has become a more reliable strategy of cryopreservation, because of its simplicity and high clinical efficiency with better clinical outcomes.


Achache H, Revel A. Endometrial receptivity markers, the journey to successful embryo implantation. Hum Reprod Update. 2006;12:731–46.CrossRefPubMed

Al-Azemi M, Kyrou D, Kolibianakis EM, Humaidan P, van Vaerenbergh I, Devroey P, Fatemi HM. Elevated progesterone during ovarian stimulation for IVF. Reprod Biomed Online. 2012;24:381–8.CrossRefPubMed

Al-Hasani S, Ozmen B, Koutlaki N, Schoepper B, Diedrich K, Schultze-Mosgau A. Three years of routine vitrification of human zygotes: is it still fair to advocate slow -rate freezing? Reprod Biomed Online. 2007;14:288–93.CrossRefPubMed

Bourgain C, Devroey P. The endometrium in stimulated cycles for IVF. Hum Reprod Update. 2003;9:515–22.CrossRefPubMed

Dehbashi S, Parsanezhad ME, Alborzi S, Zarei A. Effect of clomiphene citrate on endometrium thickness and echogenic patterns. Int J Gynaecol Obstet. 2003;80:49–53.CrossRefPubMed

Devreker F, Pogonici E, DeMaertelaer V, Revelard P, Vanden Bergh M, Englert Y. Selection of good embryos for transfer depends on embryo cohort size: implications for the ‘mild ovarian stimulation’ debate. Hum Reprod. 1999;14:3002–8.CrossRefPubMed

Devroey P, Bourgain C, Macklon NS, Fauser BC. Reproductive biology and IVF: ovarian stimulation and endometrial receptivity. Trends Endocrinol Metab. 2004;15:84–90.CrossRefPubMed

Díaz-Gimeno P, Horcajadas JA, Martínez-Conejero JA, Esteban FJ, Alama P, Pellicer A, et al. A genomic diagnostic tool for human endometrial receptivity based on the transcriptomic signature. Fertil Steril. 2011;95:50–60.CrossRefPubMed

Fauser BCJM, Devroey P, Yen SSY, et al. Minimal ovarian stimulation: appraisal of potential benefits and drawbacks. Hum Reprod. 1999;14:2681–6.CrossRefPubMed

Ferraretti AP, La Marca A, Fauser BC, Tarlatzis B, Nargund G, Gianaroli L. ESHRE consensus on the definition of ‘poor response’ to ovarian stimulation for in vitro fertilization: the bologna criteria. Hum Reprod. 2011;26:1616–24.CrossRefPubMed

Fritz MA, Holmes RT, Keenan EJ. Effect of clomiphene citrate treatment on endometrial estrogen and progesterone receptor induction in women. Am J Obstet Gynecol. 1991;165(1):177–85.CrossRefPubMed

Gandhi G, Allahbadia G, Kagalwala S, Allahbadia A, Ramesh S, Patel K, et al. ACCU-VIT: a new strategy for managing poor responders. Hum Reprod. 2013;28:i149–206.CrossRef

Gandhi G, Allahbadia G, Kagalwala S, Khatoon A, Hinduja R, Allahbadia A. IVF lite – a new strategy for managing poor ovarian responders. IVF Lite. 2014;1(1):22–8.CrossRef

Greenblatt RB, Bafrield WE, Jungck EC, Ray AW. Induction of ovulation with MRL/41. Preliminary report. JAMA. 1961;178:101–4.CrossRefPubMed

Groothuis PG, Dassen HH, Romano A, Punyadeera C. Estrogen and the endometrium: lessons learned from gene expression profiling in rodents and human. Hum Reprod Update. 2007;13:405–17.CrossRefPubMed

Hanoch J, Lavy Y, Holzer H, Hurwitz A, Simon A, Ravel A, et al. Young low responders protected from untoward effects of reduced ovarian response. Fertil Steril. 1998;69:1001–4.CrossRefPubMed

Keay SD, Liversedge NH, Mathur RS, Jenkins JM. Assisted conception following poor ovarian response to gonadotropin stimulation. Br J Obstet Gynaecol. 1997;104:521–7.CrossRefPubMed

Kolibianakis E, Bourgain C, Albano C, Osmanagaoglu K, Smitz J, Steirteghem AV, et al. Effect of ovarian stimulation with recombinant follicle-stimulating hormone, gonadotropin release hormone antagonists, and human chorionic gonadotropin on endometrial maturation on the day of oocyte pick-up. Fertil Steril. 2002;78:1025–9.CrossRefPubMed

Kolibianakis EM, Venetis CA, Tarlatzis BC. Cryopreservation of human embryos by vitrification or slow freezing: which one is better? Curr Opin Obstet Gynecol. 2009;21:270–4.CrossRefPubMed

Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril. 2002;78(3):449–54.CrossRefPubMed

Luyet B. Working hypothesis on the nature of life. Biodynamica. 1937a;1:1–7.

Luyet B. The vitrification of organic colloids and protoplasm. Biodynamica. 1937b;1:1–14.

Mandelbaum J. Embryo and oocyte cryopreservation. Hum Reprod. 2000;15:43–7.CrossRefPubMed

Martínez-Conejero JA, Simon C, Pellicer A, Horcajadas JA. Is ovarian stimulation detrimental to the endometrium? Reprod Biomed Online. 2007;15:45–50.CrossRefPubMed

Moragianni VA, Penzias AS. Cumulative live-birth rates after assisted reproductive technology. Curr Opin Obstet Gynecol. 2010;22:189–92.CrossRefPubMed

Nikas G, Develioglu OH, Toner JP, Jones Jr HW. Endometrial pinopodes indicate a shift in the window of receptivity in IVF cycles. Hum Reprod. 1999;14:787–92.CrossRefPubMed

Paulson RJ. Hormonal induction of endometrial receptivity. Fertil Steril. 2011;96:530–5.CrossRefPubMed

Paulson RJ, Sauer MV, Lobo RA. Embryo implantation after human in vitro fertilization: importance of endometrial receptivity. Fertil Steril. 1990;53:870–4.PubMed

Philips Z, Barraza-Llorens M, Posnett J. Evaluation of the relative cost-effectiveness of treatments for infertility in the UK. Hum Reprod. 2000;15:95–106.CrossRefPubMed

Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at −196 degrees C by vitrification. Nature. 1985;313(6003):573–5.CrossRefPubMed

Rezazadeh Valojerdi M, Eftekhari-Yazdi P, Karimian L, et al. Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos. J Assist Reprod Genet. 2009;26:347–54.PubMedCentralCrossRefPubMed

Roque M, Lattes K, Serra S, Sola I, Geber S, Carreras R, Checa MA. Fresh embryo transfer versus frozen embryo transfer in in vitro fertilization cycles: a systematic review and meta-analysis. Fertil Steril. 2013;99(1):156–62.CrossRefPubMed

Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C, Thomas S. High ongoing pregnancy rates after deferred transfer through bipronuclear oocyte cryopreservation and post-thaw extended culture. Fertil Steril. 2009;92:1594–9.CrossRefPubMed

Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C, Thomas S. Embryo cryopreservation rescues cycles with premature luteinization. Fertil Steril. 2010a;93:636–41.CrossRefPubMed

Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C, Thomas S. Similar ongoing pregnancy rates after blastocyst transfer in fresh donor cycles and autologous cycles using cryopreserved bipronuclear oocytes suggest similar viability of transferred blastocysts. Fertil Steril. 2010b;93:319–21.CrossRefPubMed

Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C, Thomas S. Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfer in normal responders. Fertil Steril. 2011;96:344–8.CrossRefPubMed

Simon C, Cano F, Valbuena D, Remohí J, Pellicer A. Clinical evidence for a detrimental effect on uterine receptivity of high serum estradiol levels in high and normal responders patients. Hum Reprod. 1995;10:2432–7.CrossRefPubMed

Simon C, Velasco JJG, Valbuena D, Peinado JA, Moreno C, Remohí J, et al. Increasing uterine receptivity by decreasing estradiol levels during the preimplantation period in high responders with the use of a follicle-stimulating hormone step-down regimen. Fertil Steril. 1998;70:234–9.CrossRefPubMed

Stehlik E, Stehlik J, Katayama KP, Kuwayama M, Jambor V, Brohammer R, Kato O. Reprod Biomed Online. 2005;11(1):53–7.CrossRefPubMed

Tarlatzis BC, Zepiridis L, Grimbizis G, Bontis J. Clinical management of low ovarian response to stimulation for IVF: a systematic review. Hum Reprod Update. 2003;9:61–76.CrossRefPubMed

Teramoto S, Kato O. Minimal ovarian stimulation with clomiphene citrate: a large- scale retrospective study. Reprod Biomed Online. 2007;15:134–48.CrossRefPubMed

Woods EJ, Benson JD, Agca Y, Critser JK. Fundamental cryobiology of reproductive cells and tissues. Cryobiology. 2004;48(2):146–56.CrossRefPubMed

Zhang J, Chang L, Sone Y, Silber S. Minimal ovarian stimulation (mini-IVF) for IVF utilizing vitrification and cryopreserved embryo transfer. Reprod Biomed Online. 2010;21:485–95.CrossRefPubMed