{"paper_id":"73529e85-25e0-4f04-aa6f-dd228d3d2d81","body_text":"In recent years, declining fertility rates has been observed worldwide; for example,\nin the USA, a 4% decrease in the number of live births was recorded between 2019 and\n2020 ( Eisenberg  et al .,\n2021 ). Globally, the reasons for this decline are multifactorial, although\ndelayed childbirth due to personal, professional or financial reasons may be a key\ncontributing factor in developed countries ( Mertes\n& Pennings, 2011 ). However, women who delay childbirth for whatever\nreason may face problems conceiving, owing to the age-related decline in fertility,\nas advanced maternal age (AMA) can impact oocyte and embryo competence through\nseveral mechanisms, with 35 years defined as the lowest age threshold to define AMA\n( Cimadomo  et al .,\n2018 ).\nAssisted reproductive technology (ART) may be an option for women who delay\nchildbirth to achieve their desire to build a family; however, these women may still\nhave a high chance of recurrent failed cycles ( Bashiri  et al ., 2018 ) or low prognosis for live birth\n( Schmidt  et al ., 2012 ).\nIn some cases, oocyte donation from healthy, fertile women who voluntarily undergo\novarian stimulation (OS) is an option for women who delay childbirth ( Lutjen  et al ., 1984 ;  Hammoud  et al ., 2009 ). While\nthe legality and legislative framework for oocyte donation varies considerably among\ncountries ( Calhaz-Jorge  et al .,\n2020 ;  López  et\nal ., 2021 ), current indications for women seeking oocyte\ndonation include hypergonadotropic hypogonadism, diminished ovarian reserve, having\n(or being a known or suspected carrier of) a significant genetic defect or a family\nhistory of a condition, poor oocyte and/or embryo quality or multiple previous\nfailed attempts to conceive via ART. In addition, where permitted, oocyte donation\nmay be an option for men who do not have a female partner or those who have a\ntrans-female partner and are planning to use a gestational carrier ( Lutjen  et al ., 1984 ). AMA is\nalso an indication for women seeking oocyte donation, usually due to delayed\nchildbirth and/or in conjunction with the above-mentioned indications ( Cimadomo  et al ., 2018 ).\nSeveral exogenous gonadotropin preparations are indicated for OS, including highly\npurified human menopausal gonadotropin (HP-hMG), which is purified from the urine of\npostmenopausal women and comprises FSH and LH-like activity derived from human\nchorionic gonadotropin (hCG), and recombinant human FSH (r-FSH) and LH (r-LH), which\nare produced using recombinant DNA technology ( Lunenfeld  et al ., 2019 ). The recombinant products are\navailable as separate products ( EMA, 2022a ;\n EMA, 2022b ) or as a 2:1 fixed-ratio\ncombination product ( EMA, 2023 ). In the\ngeneral population undergoing ART procedures, OS with r-hFSH:r-hLH 2:1 has been\nreported to yield a higher number of oocytes retrieved than HP-hMG ( Pacchiarotti  et al ., 2010 ;\n Fábregues  et al .,\n2013 ;  Revelli  et al .,\n2015 ;  Kirshenbaum  et\nal ., 2021 ). However, there is little published evidence on\noocyte quantity or oocyte quality following any specific treatment protocol in the\ncontext of oocyte donors. A recent systematic review and meta-analysis identified\nfive studies that evaluated the effect of the type of gonadotropin used for OS in\noocyte donors ( Martinez  et al .,\n2021 ). Three of these studies compared HP-FSH  versus  hMG\n( Söderström-Anttila  et\nal ., 1996 ), r-hFSH  versus  r-hFSH + hMG\n( Tesarik  et al ., 2002 )\nor r-hFSH  versus  HP-hMG  versus  r-hFSH + HP-hMG\n( Melo  et al ., 2010 ),\nrespectively, in a long agonist protocol; and two studies compared r-hFSH\n versus  r-hFSH + r-hLH ( Acevedo\n et al ., 2006 ) or corifollitropin alfa\n versus  hMG ( Cruz  et\nal ., 2017 ), respectively, in a gonadotropin releasing\nhormone (GnRH) antagonist protocol. Although no difference in the number of oocytes\nretrieved was reported between the studies using GnRH antagonists versus agonists,\nno further conclusions could be drawn owing to the high degree of heterogeneity\namong the studies.\nAs an alternative to using GnRH analogs to prevent the premature luteinizing hormone\n(LH) surge during OS, recent work has focussed on the use of stimulation cycles\nusing progesterone (progestin-primed OS [PPOS]) to prevent early luteinisation\n( La Marca & Capuzzo, 2019 ;  Glujovsky  et al ., 2023 ). PPOS\nprotocols are advantageous in that they can be administered orally, are flexible and\neconomic for patients, and control over the LH surge is achieved rapidly ( Kuang  et al . 2015 ;  Qin  et al ., 2016 ,  Massin, 2017 ). These factors highlight the\npotential of PPOS to be a cost-effective and patient-friendly oral alternative to\ninjected GnRH analogs in younger women, whose motivation for oocyte donation is\nusually solely altruistic and, therefore, presents unique challenges for physicians\nwhen balancing the risk and benefits to ensure the wellbeing and convenience of the\ndonor ( Kool  et al .,\n2018 ).\nRecent studies have demonstrated few or no differences between PPOS and a GnRH\nantagonist protocols in oocyte quality ( Yildiz\n et al ., 2019 ) or competence ( Vaiarelli  et al ., 2024 ), oocyte and MII oocyte\nnumber and number of cleavage and blastocyst embryos ( Duc Thang  et al ., 2024 ), oocyte maturation\nrates and trigger-to-oocyte retrieval interval ( Ata\n et al ., 2024 ). However, there is no consensus on\nwhich populations of women may most benefit from PPOS (e.g., poor responders, women\nwith endometriosis, women of advanced maternal age, young women for oocyte donation\nprocedures) ( Glujovsky  et al .,\n2023 ). Furthermore, there are no robust data on any potential interaction\nbetween supraphysiological levels of progesterone in PPOS and the different LH-like\nactivity in the commonly used preparations, and the effect this may have on oocyte\nquality.\nThe objective of the present study was to investigate the number of good-quality\nblastocysts obtained from population of young women treated in the late follicular\nphase with r-FSH:r-LH in a 2:1 fixed ratio combination or HP-hMG, following 6 days\nof treatment with r-FSH in a PPOS protocol in a population of young women.\n\nThe aim of this pilot randomized controlled trial was to compare the blastocyst\nmorphology of donor oocytes following OS with r-FSH:r-LH in a 2:1 fixed ratio\ncombination (Pergoveris; Merck KGaA, Darmstadt, Germany) or HP-hMG (Menopur;\nFerring, West Drayton, UK), both starting from stimulation Day 7 following 6\ndays of stimulation with r-FSH alfa (Gonal-f; Merck KGaA, Darmstadt, Germany).\nThe study was conducted from August 2022 to June 2023 at a single centre in\nLima, Peru. The study design is shown in  Figure\n1 .\nFigure 1 Study design.\nStudy design.\nWomen undergoing OS for oocyte donation were screened under informed consent for\ninclusion and exclusion criteria through blood testing, and general medical\nevaluation.\nWomen were eligible as oocyte donors if they met all the following criteria: age\n19-30 years at the time of providing informed consent; recorded levels of AMH\n≥2 ng/mL, and BMI 21.0-29.9 kg/m 2 . All donors underwent\nphysical, gynecological and psychological examinations. Additionally, they\nshould be no family history of hereditary diseases or chromosomal disorders. All\nparticipants had a normal karyotype, and tested negative for sexually\ntransmitted diseases and toxicological screening, and had an up-to-date negative\ncervical cancer screening. Recruitment of oocyte donors was done based on\nrecommendations given by other donors and the reason for gamete donation was\nsolely altruistic.\nSperm was obtained from the partners of the oocyte recipients (homologous) or\nfrom sperm donors (heterologous) in the case of poor semen quality ( Boitrelle  et al ., 2021 ).\nMen aged 21-50 years were eligible as sperm donors if they met the criteria for\ndonation at the treating clinic or another certified cryobank. Sperm quality was\nassessed according to semen volume, and sperm motility, quantity and\nmorphology.\nParticipants were randomized (1:1) using a computer-generated algorithm to either\nGroup 1 (r-hFSH:r-hLH 2:1 fixed ratio combination) or Group 2 (HP-hMG) before\nreceiving the first injection for OS. No blinding was performed.\nAll donors were stimulated with 225 IU/day r-FSH (Gonal-f, Merck KGaA, Darmstadt,\nGermany) for the first six days. All donors received 200 mg/day oral micronized\nprogesterone from Day 1 of stimulation (PPOS). From Day 7 onwards, oocyte donors\nin Group 1 were treated with 225 IU/day r-hFHS:r-hLH 2:1 fixed ratio combination\n(150 IU r-FSH: 75 IU r-hLH), and oocyte donors in Group 2 were treated with 225\nIU/day HP-hMG ( Figure 1 ).\nFollicular development was monitored by serial ultrasound scans. The first scan\nwas conducted at the beginning of the cycle, with the second scan after 5 days\nof gonadotropin treatment, and then every 2 days until follicle size reached 16\nmm; a final scan was conducted on the day of ovarian maturation trigger, when\ntwo follicles had reached a mean diameter ≥17 mm. Oocyte maturation was\ntriggered using 0.2 mg triptorelin, with oocyte pick-up at 34-36 hours after\ntriggering.\nHomologous sperm was obtained from the recipient’s partner in accordance with\nclinic protocols. Heterologous sperm was collected from the cryobank local to\nthe treating centre or from another certified cryobank, and then transferred to\nthe clinic as a frozen specimen in accordance with clinic standards. All\ninseminations were performed using intracytoplasmic sperm injection (ICSI).\nZygotes were cultured individually until day 5/6 in drops of 25 µL of\nGlobal Total® media (Lifeglobal, Canada) under mineral oil at 37 °C in 7%\nCO 2 , 5% O 2  and 88% N 2  (adapted from  Sepúlveda  et al .,\n2009 ). Embryos were classified according to the Society for Assisted\nReproductive Technology (SART) classification ( Racowsky  et al ., 2010 ).\nEmbryos were vitrified using a modified Cryotop method ( Kuwayama  et al ., 2005 ). Embryos were\nequilibrated at room temperature for 12 minutes in the equilibrium solution\n(TCM-199 medium/20% synthetic serum substitute/7.5% (v/v) ethylene glycol/7.5%\ndimethyl sulfoxide). Embryos were then transferred to the vitrification solution\n(TCM-199 medium/20% synthetic serum substitute/15% ethylene glycol/15% dimethyl\nsulfoxide/0.5 M sucrose) and incubated for 1 minute before being placed in a\ncryoslip (one embryo per strip) and immediately submerged in liquid\nnitrogen.\nThe primary outcome was the number of blastocysts with good morphology. All\nembryos were evaluated by two senior embryologists using the SART\nclassification ( Racowsky  et\nal ., 2010 ) and categorized as either good, fair or\npoor: a good grading was assigned to embryos with Grade A inner cell mass\nand trophectoderm Grade A or B (AA or AB blastocysts); a fair grading was\nassigned to embryos with Grade B inner cell mass and trophectoderm Grade A,\nB or C (BB, BC or BA blastocysts); a poor grading was assigned to embryos\nwith Grade C inner cell mass (CC or CB blastocysts).\nSecondary outcomes were the total number of oocytes recovered and\ninseminated, the number of 2PN embryos, the total number of blastocysts, and\nthe proportion of good-quality blastocysts, calculated as the proportion of\nembryos to be of good quality over all embryos assessed.\nAll donors were monitored for adverse events during OS, and were asked to\nself-report any adverse events occurring within two weeks after oocyte\nretrieval. The potential risks associated with the study were those common\nto all ART procedures: the risks of OS were ovarian hyperstimulation\nsyndrome (OHSS) and ovarian torsion; the risks of egg retrieval were\nbleeding, risks associated with anaesthesia, infection, and trauma.\nMonitoring involved regular transvaginal ultrasound scans and hormonal blood\ntests to monitor follicular development and ovarian response. Ultrasound\nscans were performed as previously described. Donors were scheduled for a\nfollow-up clinical evaluation throughout the two weeks after oocyte\nretrieval to assess recovery and detect any delayed adverse events. Any\nadverse events reported by donors were to be documented in real time by the\nclinical team and coded using the Medical Dictionary for Regulatory\nActivities (MedDRA). The data collection process adhered to established\nclinical protocols and ethical guidelines, ensuring donor safety and\ncompliance with research standards.\nWe estimated that 80 participants would be required to obtain 80% power to\ndemonstrate a difference of at least one blastocyst in the number of\ngood-morphology blastocysts, assuming a mean number of blastocysts in the HP-hMG\ngroup of 4.9, based on our previous observations and assuming a loss rate of\n10%. Between-group differences in blastocyst morphology were assessed using the\nKruskal-Wallis test. All other analyses were descriptive.\nThis study protocol was approved by the local Ethics Committee. All participants\nprovided informed consent after counselling. Consent was obtained by the\ninvestigator or a member of their team directly from the participants before\nthat subject takes part in the study. Consent was documented either with a\nhandwritten signature on paper or through an electronic signature following the\nGuidance on Use of Electronic Informed Consent, according to the patient’s\npreference. All participants received a copy of the Informed Consent Form.\n\nOf the 80 donors recruited, seventy-nine were included in the analysis. One\ndonor in Group 1 was excluded after their OS was cancelled. The baseline\ncharacteristics were similar between the women randomly assigned to either\ngroup ( Table 1 ).\nBaseline characteristics and stimulation results for the oocyte\ndonors.\nThe mean paternal age was 36 (SD 10) years in Group 1 and 37 (SD 12) years in\nGroup 2. Most semen samples were homologous (Group 1: 72.0%; Group 2: 27.7%)\ncompared with heterologous (Group 1: 28.8%; Group 2: 27.8%) and the highest\nproportion of inseminations used fresh semen (Group 1: 48.4%; Group 2: 53.3%)\nrather than frozen semen (Group 1: 51.6%; Group 2: 46.7%).\nThe total FSH dose was 2140 (SD 205) IU in Group 1 and 2120 (SD 227) IU in Group\n2. The length of stimulation was 9.5 (SD 0.9) days in Group 1 and 9.4 (SD 1.0)\ndays in Group 2 ( Table 1 ). The mean\nnumber of oocytes retrieved was 22.8 (SD 10.4) in Group 1 and 22.4 (SD 10.3) in\nGroup 2 ( Table 1 ).\nOverall, 97 embryo transfers were registered, 50 in Group A and 47 in Group\nB. The mean (SD) number of good-quality blastocysts was statistically\nsignificantly higher in the women in Group 1 compared with those in Group 2\n(3.4 [2.0]  vs . 2.3 [2.0]; difference 0.9 [95% CI 0.6 to\n1.7]; Kruskal-Wallis test  p =0.0359) ( Table 2 ). There were no differences in the number of\nfair-quality (0.4 [95%CI -0.18 to 1.0]) or poor-quality (0.5 [95% CI -0.4 to\n0.3]) blastocysts between the treatment groups.\nResults of ovarian stimulation per stimulation cycle following\nICSI\nBlastocyst morphology assessed according to the Gardner and SART\ngrading systems.\nGroup 1 had a higher total number of blastocysts compared with Group 2\n(difference 1.4 [95% CI 0.3 to 2.4]) ( Table\n2 ). There were no differences in the total number of oocytes\ninseminated (difference 0.3 [95% CI -0.4 to 1.0]), the total number of 2PN\nembryos (difference 0.2 [95% CI -0.7 to 1.1]), or the proportion of\ngood-quality blastocysts (difference 8.2% [95% CI -20.0% to 25.6%]) between\ntreatment groups ( Table 2 ). Finally,\nongoing pregnancy rates were similar among the group (62.5% for Group 1 and\n58.9% for Group).\nNo adverse events were identified during the study. There were no reports of\nOHSS, ovarian torsion or other procedure-related complications.\n\nThe results of this randomized pilot study show that OS with r-FSH:r-LH in a 2:1\nfixed ratio combination from Day 7 resulted in a significantly higher number of\ngood-quality blastocysts compared with HP-hMG from Day 7 in oocyte donors using a\nPPOS protocol to prevent premature LH surge. These preliminary results using PPOS in\nhealthy, young women show a potentially lower oocyte developmental competence when\nurinary gonadotrophins are used in combination with follicular phase progesterone in\nthis population.\nPremature LH surge can compromise oocyte yield in\n in - vitro  fertilization (IVF)/ICSI and is one\nof the major reasons for cycle cancellation in OS ( Messinis, 2006 ). However, the use of GnRH analogs (either GnRH agonists\nor GnRH antagonists ( Macklon  et\nal ., 2006 )) have recognised drawbacks. GnRH agonists increase\nthe risk of OHSS through the requirement for hCG triggering, whereas GnRH\nantagonists can have variable effectiveness in some patient populations, including\nolder patients and those with diminished ovarian reserve ( Bosch  et al ., 2003 ;  Reichman  et al ., 2014 ). Furthermore, both GnRH\nagonists and antagonists are injectable products, which increases the cost,\ncomplexity and patient burden of OS.\nStudies using progesterone in the follicular phase have shown that it is a regulator\nof ovulation timing and a potentially convenient and flexible oral alternative to\nGnRH agonists for preventing early luteinization when used in a PPOS protocol in\nfreeze-all cycles ( Kuang  et al .,\n2015 ). Most of the studies on PPOS published to date have compared the\noutcomes GnRH analog protocols and PPOS using locally available preparations of hMG\nfor OS, although there are a few studies that directly compare the outcomes from\ndifferent gonadotropin preparations. In a retrospective study covering two discrete\ntime periods (2019 and 2020), during which two different OS regimens were used\n(2019: corifollitropin, recombinant FSH and GnRH antagonists, with ovulation\ntriggered using GnRH agonist; 2020: PPOS protocol with human menopausal gonadotropin\n[hMG] and dual trigger [GnRH agonist and low dose hCG]), there was no difference in\nthe ratio of the number of mature oocytes and serum AMH between the two periods (4.0\n[2.3-7.1]  vs . 4.0 [2.7-6.8];  p =0.80), although the\ntotal cost of drugs used for OS was lower in the second period €940 [€774-€1,096]\n vs . €520 [€434-€564];  p <0.001) ( Filippi  et al ., 2023 ). In\naddition, a retrospective study in women who were stimulated with r-FSH (Gonal-f,\nMerck KGaA, Germany) or HP-hMG (Merional, IBSA Institut, Switzerland) in a PPOS\nprotocol (Dydrogesterone [Duphaston® 10 mg, Abbott, Turkey]) reported that\nthe total number of follicles, oocytes, MII and 2PN oocytes, fertilization rates and\npregnancy rates were similar between the two groups, although higher doses of\ngonadotropin were used in the HP-hMG group compared with the r-FSH group\n( p =0.001) ( Yetkinel  et\nal ., 2024 ). While these early results may demonstrate the\npromise of using PPOS cycles, they do not address any potential differences in the\ngonadotropin preparations that are commonly used in OS, specifically the FSH and LH\nactivity in r-FSH:r-hLH 2:1, which are produced using complementary DNA technology\nand have a high level of purity and batch-to-batch consistency ( Bassett & Driebergen, 2005 ;  Lispi  et al ., 2006 ;  Bassett  et al ., 2009 ;  Lispi  et al ., 2023 ) or the\nLH-like activity in HP-hMG, which is mainly derived from hCG purified from pregnant\nwomen ( Capolupo  et al .,\n2024 ). Furthermore, there are no studies that investigate how these\ncomponents may interact with the supraphysiological concentrations of progesterone\nused in PPOS.\nAlthough LH and hCG share approximately 85% structural identity and bind to the same\nreceptor (LHCGR) on the antral follicles, they cannot be considered equivalent, as\nthey interact differently with the receptor and activate different cellular pathways\n( Casarini  et al ., 2018 ).\nThe action of LH is preferentially exerted through phosphorylated\nextracellular-regulated kinase 1/2 (pERK1/2) and phosphorylated AKT (also known as\nprotein kinase B), resulting in proliferative/antiapoptotic signals and partial\nagonism of progesterone production  in vitro . In contrast, hCG\ndisplays notable cAMP/protein kinase A (PKA)-mediated steroidogenic and proapoptotic\npotential ( Casarini  et al .,\n2018 ). Therefore, on the one hand, r-hLH may have a positive effect on\noocyte maturation, potentiated by r-FSH, as well as having an anti-apoptotic effect\non cumulus cells and promoting the paracrine signalling required for cell expansion\nand oocyte signalling during folliculogenesis ( Ruvolo  et al ., 2007 ;  Huang  et al ., 2015 ). On the other hand, the hCG in\nHP-hMG may have a detrimental effect on oocyte development by activating the\nsteroidogenic and proapoptotic pathways.\nProgesterone contributes to normal mammalian ovarian function and has several\ncritical functions during embryo development and implantation, including endometrial\nreceptivity, embryonic survival during gestation, and transformation of the\nendometrial stromal cells to decidual cells ( Salehnia & Zavareh, 2013 ). While hCG has greater steroidogenic\npotential than LH, reflecting its role during pregnancy to support increased\nprogesterone production, the dual proliferative and androgen-stimulating roles\nexerted by LH in granulosa and theca cells, respectively, during folliculogenesis\nmight not require induction of the steroidogenic pathway at the levels needed in\npregnancy ( Casarini  et al .,\n2018 ). Furthermore, high levels of progesterone, such as the\nsupraphysiological levels due to PPOS, may even have local doseand species-dependent\neffects on the developing oocyte ( diZerega &\nHodgen, 1982 ;  Kim & Greenwald,\n1987 ;  Setty & Mills, 1987 ;\n Komatsu & Masubuchi, 2017 ). In a\nretrospective analysis of 1200 cycles of women stimulated with hMG in either a PPOS\nor GnRH antagonist protocol, at similar cumulative hMG doses, the number of\nfollicles with diameter >10mm, the number of retrieved oocytes and the number of\nMII oocytes were lower in the PPOS protocol compared with the antagonist protocol,\nalthough the oocyte yields were rescued by higher doses of hMG ( Long  et al ., 2021 ). These\nexploratory results we report here add weight to these preliminary data on the\npotential detrimental effect of the LH-like activity of hCG in conjunction with high\nlevels of progesterone from PPOS, although this needs further investigation.\nWhile we report a higher number of good-quality blastocysts, we did not observe a\ndifference in the number of oocytes retrieved, which is contrary to the results of\nother studies that compare these two formulations. In a meta-analysis of six studies\nof OS with r-FSH:r-hLH 2:1 fixed ratio combination versus HP-hMG, a higher mean\nnumber of oocytes was retrieved in the rFSH:r-LH 2:1 fixed ratio combination cohort\n(1.43 [95% CI 0.18, 2.69];  p =0.025) ( Dahan  et al ., 2026 ). However, the studies\nanalysed reported on women undergoing routine IVF cycles using autologous oocytes,\nand there are few published data on the use of these treatments in donor cycles. In\none study of donor cycles, treatment with r-FSH:r-hLH in a 2:1 ratio also resulted\nin a higher number of oocytes retrieved (16.5 [SD 4.1]  vs . 11.8 [SD\n2.6];  p =0.049) compared with treatment with urinary gonadotropins\n(HP-hMG plus uFSH) ( Requena  et al .,\n2014 ). This variance from the published literature warrants further\ninvestigation into the outcomes of different OS protocols specifically in donor\ncycles.\nThe strengths of this trial are it was a randomized pilot trial conducted at a single\ncentre that recruited a representative population of women participating in oocyte\ndonation. Some limitations also have to be acknowledged. Randomized treatment only\ncommenced after stimulation Day 6, prior to which both cohorts were treated with\nr-FSH; therefore, the effect of the test compounds from Day 1 of stimulation could\nnot be addressed, and no sperm DNA fragmentation was performed, also comparing the\noutcome to stimulation with rFSH alone.\n\nIn this randomized controlled pilot study we show that late follicular phase\ntreatment with r-FSH:r-LH in a 2:1 fixed ratio combination resulted in a\nsignificantly higher number of good-quality blastocysts compared with late\nfollicular phase HP-hMG following OS with r-FSH until Day 6 in oocyte donors. While\nthese data need to be confirmed in larger clinical studies, optimizing oocyte\nquality in PPOS cycles may improve the chances of live birth resulting from a single\ncycle, thus reducing the burden on oocyte donors.","source_license":"CC-BY-4.0","license_restricted":false}