Clinical strategies for ART treatment of infertile women with advanced maternal age

Abstract Background An ever‐increasing number of women in our country with advanced maternal age are choosing to achieve pregnancy. This means effective strategies are needed for infertile patients. Questions arise, however, concerning the need for ovarian stimulation, and, if so, whether intracytoplasmic sperm injection (ICSI) is better than conventional insemination for those women who may have only one mature oocyte. Methods We evaluated our data to answer these questions. Herein, we also introduce our strategy for patients who show unsynchronized follicular growth. Main findings Ovarian stimulation in ART treatment for patients with advanced maternal age has resulted in the achievement of higher pregnancy rates, and therefore, this form of stimulation is often selected. Based on our data, ICSI as an insemination procedure has not improved clinical pregnancy rates compared with conventional insemination and has actually decreased the clinical pregnancy rates. Conclusion In this article, we reviewed and compared the protocols and strategies that are available to increase the number of developed embryos for the patients with advanced maternal age. We hope that this review will be helpful for both patients and clinicians.

and 44 years of age found it quite difficult to conceive naturally or even via the use of intrauterine insemination. ART and IVF treatments now play an important role in the field of infertility treatment and have made a significant contribution to the increased number of newborns. The number of infertile patients with advanced maternal age (≥40 years) has seen a recent increase. Even with the increased use of ART treatment, however, the pregnancy rates are not yet satisfactory among patients who are more than 40 years of age. The miscarriage rate, unfortunately, has increased due to these increases in the advanced maternal age. 4 Indeed, there were 6658 cycles planned for oocyte pickup (OPU) for ART treatment at Sugiyama Clinic between January and December in 2016, and 48.1% (3201 cycles) of them were for patients who were more than 40 years of age. This recent trend is not exclusive to our clinic, but in fact is becoming common in Japan.
The clinicians working at the Department of Reproductive Medicine and in clinics that specialize in infertility treatment encounter many patients among these age-groups every day. Even though the success rate of IVF treatment for these patients is relatively low even when using ART treatment, these may be the only viable options for infertile patients with advanced maternal age. When devising strategies to overcome the poor success rate, two main questions often must be answered: one is that whether ovarian stimulation is needed or not, and the other is whether intracytoplasmic sperm injection (ICSI) be better than conventional insemination as an insemination method for patients who may have only one mature oocyte? In this article, we expanded on and discuss these two main points. In addition, our strategy for the patients showing unsynchronized follicular growth is also introduced.

| IS OVARIAN S TIMUL ATI ON NEEDED FOR THE PATIENTS WITH ADVAN CED MATERNAL AG E?
Between 2012 and 2016, a total of 3545 cycles were recruited for analysis. The patients recruited in these treatment cycles satisfied the following two conditions: They were more than 40 years of age at the time of OPU and they had used OPU <2 times. Moreover, all patients had received fresh embryo transfers, and patients receiving cryopreserved embryos were excluded, because these embryos did not always reflect the maternal age (cryopreservation of embryos could make the difference of the patients' age between the cryopreservation day and embryo transfer day). All cycles were divided into one of two groups: the stimulation group and the minimal stimulation group. There were 1666 cycles in the stimulation group, and this group received ovarian stimulation that included either the gonadotropin-releasing hormone (GnRH)-agonist long protocol 5 or the GnRH-agonist short protocol, 6 along with either clomiphene citrate (CC) only or a combination of CC and recombinant follicle-stimulating hormone (FSH). 7 There were 1879 cycles in the minimal stimulation group, which used only CC or letrozole, or a natural ovulatory cycle. To calculate and evaluate the clinical pregnancy rate, OPU cycles were used rather than embryo transfer cycles. Patients and clinicians alike usually act as an index to evaluate the effectiveness of ART. However, this rate only indicates the number of patients who were able to achieve pregnancy by receiving embryo transfer, which does not reflect the number receiving OPU. This rate does not include the number of patients who received OPU without an embryo for embryo transfer. Patients often inquire about the success rate for OPU. Therefore, the pregnancy rate per OPU cycle was selected to evaluation. Clinical pregnancy was defined as confirmation via vaginal ultrasound of the existence of a gestational sac in the uterine cavity.
The ART outcomes for the two groups are listed in Table 2 patients in the stimulation group, 7.1% (n = 118) of total cycles resulted in no usable oocytes for the following reasons: cancelation of OPU for just after ovulation (n = 48), no oocyte to retrieve (n = 26), or retrieval of a degenerated oocyte (n = 44). By contrast, the percentage of patients who could obtain no usable oocytes in the minimal stimulation group was 49.0% (n = 920), which was significantly higher than that in the stimulation group (P < 0.01). The reasons were the same: n = 580, n = 142, and n = 198, respectively. In the stimulation group, the OPU cancelation rate was 2.8%, which was significantly lower than that in the minimal stimulation group (30.9%, P < 0.01). In the stimulation group, patients received some agents to prevent ovulation such as GnRH-agonist, GnRH-antagonist, or CC, 8 and this was one of the reasons for the lower cancelation rate in the stimulation group. The use of GnRH-agonist and GnRH-antagonist during ovarian stimulation could suppress LH secretion from the pituitary gland and consequently could prevent ovulation; by contrast, CC acts antagonistically to the estradiol receptor at the hypothalamus level, inhibiting both negative and positive feedbacks, and resulting in the suppression of ovulation during ovarian stimulation. 8 The average number of retrieved oocytes in the stimulation group was 4.2 ± 1.8, which was significantly higher than that in the minimal stimulation group (1.2 ± 0.3, P < 0.01). In the stimulation group, 1285 cycles of fresh embryo transfers were performed, and 164 patients achieved pregnancy for a clinical pregnancy rate per OPU of 9.8%.
On the other hand, in the minimal stimulation group, at 543 cycles fresh ET was performed, and 59 pregnancies were achieved. The clinical pregnancy rate per OPU was 3.1%, which was significantly lower than for the stimulation group ( Figure 1; P < 0.01). It was considered that two reasons might cause this result. One was that a higher number of retrieved oocytes were collected in the stimulation group compared with that in the minimal stimulation group. The other was that the rate of unusable oocytes in the stimulation group was lower compared with that in the minimal stimulation group. The half of the cycles scheduled of oocyte retrieval in the minimal stimulation group could not proceed to the further step to fertilize. As a consequence, ovarian stimulation for the patients with advanced maternal age in ART treatment resulted in the achievement of a higher pregnancy rate; therefore, ovarian stimulation is recommended for these types of patients who receive ART treatment.
The only nearly 10% of the patients in the stimulation group could not obtain usable oocytes in spite of receiving ovarian stimulation, but the physicians with reproduction should suggest further plans or protocols for them. The further plans were selected according to the AMH values or basal FSH level of the patients. When the patients showed low AMH value (<1.0 ng/mL) or elevated basal FSH level (≥15 IU/L), we usually recommend them to change past protocol to minimal ovarian stimulation protocol using only CC or letrozole for their further stimulation. Because these patients with diminished ovarian reserve will not be expected to get more numbers of the oocytes even though strong stimulation using GnRH-agonist long protocol, short protocol or GnRH-antagonist protocol, except only increasing their economical and physical burden. By contrast, when the patients showed moderate AMH value (≥1.0 ng/mL) or normal basal FSH (<15 IU/L) who showed inadequate ovarian response stimulated by the conventional stimulation protocol, we will select same ovarian protocol for their next treatment.

| IS I C S I NEEDED FOR THE PATIENTS WITH ADVAN CED MATERNAL AG E WHO C AN OBTAIN ONLY ONE OO C Y TE?
Clinicians sometimes face a situation whereby patients have only one follicle on the day of maturation trigger with or without ovarian stimulation, particularly those with advanced maternal age. Within the time period and recruited cycles mentioned above, the number of cycles that showed only one follicle for oocyte retrieval in the stimulation and minimal stimulation groups was 338 and 1791, respectively. In the stimulation group, at 107 cycles embryo transfer was performed, and six pregnancies were achieved. The clinical pregnancy rate per OPU and ET was 1.9% and 17.8%, respectively.
By contrast, in the minimal stimulation group, 39 pregnancies by the 385 transfers were achieved for clinical pregnancy rates per OPU and ET of 2.3% and 10.1%, respectively (Table 3). There were no significant differences between the two groups. Based on these data, ovarian stimulation would not have increased the clinical pregnancy rate for patients who showed only one developed follicle in reaction to the ovarian stimulation.
When clinicians must choose an insemination procedure for the patients with only one oocyte, the option is either ICSI or conventional insemination. Based on Japanese ART data, the ICSI cycle is more involved than a conventional insemination cycle, and this difference has grown for each of the past 3 years. 4 This trend seems to be a worldwide phenomenon. In general, the fertilization rate of ICSI is thought to be similar to that of conventional insemination. 9,10 However, many patients and clinicians fervently believe that ICSI might be superior to conventional insemination with regard to F I G U R E 1 This graph indicates the clinical pregnancy rates per oocyte pickup in the stimulation and minimal stimulation groups. The rate in the stimulation group was 9.8% and was significantly higher than that in the minimal stimulation group (3.1%, P < 0.01) fertilization, because ICSI directly injects spermatozoa to the oocyte. For this reason, it is not uncommon that ICSI is selected as the insemination procedure for the patients who have only one oocyte.
Recently, we reported that the insemination procedure (conventional insemination or ICSI) had no influence on the outcome of ART treatment, and collecting only one oocyte is not an indication for ICSI. 11 Therefore, for the patients who could obtain only one oocyte, we compared the clinical pregnancy rate by ICSI-derived embryo with that by conventional insemination-derived embryo to evaluate which was more productive. The clinical pregnancy rate per ET derived from conventional insemination was 12.6%, which was significantly higher than that derived from ICSI (5.2%, P < 0.05; Figure 2).
Based on our data, compared with conventional insemination, ICSI did not improve the clinical pregnancy rate. Previously during ART treatment, if a patient's serum progesterone (P4) concentration was elevated (≥1.5 ng/mL) on the day maturation was triggered, OPU was usually canceled. This was the policy because an elevation of P4 reduces the rate of implantation , 12,13 which could affect oocyte quality. 14 In a recent effort to avoid fresh embryo transfer, we reported that P4 elevation (≥1.5 ng/mL) on the day of maturation trigger could not decrease the pregnancy rate. 15 In the present study, moreover, we found that P4 elevation affected neither oocyte recovery rate nor embryo development rate. 15 On the other hand, the effectiveness of randomly beginning a protocol of ovarian stimulation has been reported. 16

| DOUB LE OO C Y TE PI CKUP FOR THE PATIENTS WITH DOR AND S HOWING UNSYN CHRONIZED FOLLICUL AR G ROW TH DURING ART TRE ATMENT
The clinical pregnancy rate per ET derived from the conventional insemination was 12.6%, which was significantly higher than that derived from intracytoplasmic sperm injection (5.2%, P < 0.05) was analyzed for DOR patients ≥40 years of age who showed unsynchronized follicular growth, and the outcome of DOR was analyzed.

| A PROTO COL OF DOUB LE OPU AND THE OUTCOME OF THIS PRO CEDURE
The protocol for double OPU is shown in Figure 3  and 25.0%, respectively. Embryos were defined as MGQ embryos when they had developed to at least 6-cell stage with <20% fragmentation. 18 This system of embryo assessment was based on the classification system described by Veeck. 19 All embryos were not the first and second rounds of OPU, the usable embryos for cryopreservation numbered 10 and 5, respectively, and as a consequence, 12 of the patients were able to obtain cryopreserved embryos. Of these 12 patients, four were unable to obtain cryopreserved embryos without a second round of OPU.
Progesterone elevation on the day of the maturation trigger neither decreased the pregnancy rate when a fresh embryo transfer was avoided, nor affected the embryo development rate.
Furthermore, a random start was reported for ovarian stimulation. 16,17 This indicated that the start of ovarian stimulation was not related to the menstrual cycle. This random-start protocol has now been adapted for the patients who immediately face a surgical procedure, chemotherapy, or radiation therapy due to cancer, and ovarian stimulation can be started any time with or without an elevation of progesterone concentration. F I G U R E 4 At the first oocyte pickup (OPU), 12 out of 13 patients received oocytes, and 11 out of 13 received oocytes at the second OPU. Four patients (Patients 6, 7, 10, and 13) were able to cryopreserve embryos derived from the second OPU. Among them, a pregnancy was confirmed for Patient 4. 〇: oocytes that could be cryopreserved; : retrieved oocytes on the first OPU; : retrieved oocytes on the second OPU doubt that the transfer of euploid embryos or blastocysts is the most effective strategy for these women to achieve pregnancy, but the preimplantation genetic testing for aneuploidy (PGTA) is not allowed in our country. Moreover, the number of retrieved oocytes or developed embryos among the patients with advanced maternal age would likely be insufficient for PGTA due to either poor ovarian response or diminished ovarian reserve. In this article, we reviewed and compared the protocols and strategies that are available to increase the number of developed embryos.
We hope that this review will be helpful for both patients and clinicians.

H UMAN RI G HTS S TATEMENTS AND INFORMED CONS ENT
We obtained a written informed consent from each participant couple. This study was approved by the ethical committee of Sugiyama Clinic.

APPROVAL OF E THI C S COMMIT TEE
All the procedures were followed in accordance with the ethical standards of the responsible committee of Sugiyama Clinic and with the Helsinki Declaration of 1964 and its later amendments.

CLINI C AL TRIAL REG IS TRY
This article was not applicable to clinical trial registry.